Yeast screens for treatment of human disease

ABSTRACT

Screening methods for identifying substances that provide therapeutic value for various diseases associated with protein misfolding are provided. Genetic and chemical screening methods are provided using a yeast system. The methods of the invention provide a rapid and cost-effective method to screen for compounds that prevent protein misfolding and/or protein fibril formation and/or protein aggregation which includes numerous neurodegenerative diseases including Parkinson&#39;s disease, Alzheimer&#39;s disease, Huntington&#39;s disease as well as non-neuronal diseases such as type 2 diabetes.

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/269,157 filed on Feb. 15, 2001, which is incorporated byreference in its entirety herein.

The government may own rights in the present invention pursuant to grantnumber R37GM25874 from the National Institutes of Health. Funding fromthe Howard Hughes Medical Institute is also acknowledged.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of genetic andchemical screening and diseases associated with protein misfolding. Moreparticularly, it concerns the development of a yeast-based system thatcan be used to screen for substances that provide therapeutic value forvarious diseases associated with protein misfolding. Methods forperforming genetic and chemical screens using the yeast systems of theinvention are also provided. One major class of diseases benefited bythe methods of the invention are the neurodegenerative diseasesincluding Parkinson's disease, Alzheimer's disease, Huntington's diseaseand the like.

2. Description of Related Art

The correct folding of a protein is a key event to attain properbiological function. Correct folding leads to the characteristicconformation of a protein which determines protein activity,aggregation, degradation, and function. Several proteins are implicatedin neurodegenerative diseases, such as Parkinson's disease (PD),transmissible spongiform encephalopathies (TSEs), Alzheimer's disease(AD), familial amyloid polyneuropathy (FAP), prion diseases, andHuntington's disease (HD), among several others. These proteins formabnormal aggregates due to alternative folding mechanisms. Thesemisfolded protein aggregates form insoluble fibrils which are thendeposited in tissues. Fibrillogenesis is the cause of variouspathologies involving neuronal degeneration. Deposition of insolublefibrils in tissues leads to formation of plaques and tangles andeventual cellular degeneration as the pathology progresses. Despite alack of amino acid sequence homology of the fibril forming proteins, thefibrils have several common morphological features. For example, somecommon morphological features of amyloid fibers, (formed by amyloidproteins), include a cross β-structure, similar sizes, display of greenbirrefringence upon staining with congo red when observed underpolarized light, Thioflavin T binding, etc.

One example of a disease based on fibrillogenesis, is the pathology ofamyloidosis which is defined by the deposition of amyloid fibrils intotissues and is typified by Alzheimer's Disease (AD). Systemicamyloidosis are characterized by amyloid deposition throughout theviscera. Animal amyloid is a complex material composed partly of proteinfibrils. The protein that comprises these fibrils varies from disease todisease. β-Amyloid is one of these proteins which is involved in thepathological progression of AD.

In the case of Parkinson's disease (PD), dopaminergic neurons in thebrain undergo selective neurodegeneration. A highly conservedpre-synaptic protein, α-synuclein, with unknown function has beenimplicated in PD. Two different point mutations in α-synuclein, A53T andA30P, are involved in autosomal dominant familial PD. It is likely thatconformational changes in α-synuclein lead to the typical proteinaceousaccumulation and fibrillogenesis characteristic of such diseases.Purified full-length α-synuclein can form fibrils similar to those foundin Lewy Bodies (cytosolic inclusions) in PD. The mechanism offibrillogenesis has not been described, although recent data indicatethat α-synuclein aggregation follows a nucleation-elongation mechanism,as suggested for the other disease-related proteins.

It is well recognized in the art, that once fibrilloid deposits haveformed, there is no known therapy or treatment which significantlydissolves such deposits in situ (U.S. Pat. No. 5,643,562). Consequently,strategies based on prevention of protein aggregation and fibrilformation is a major goal in the therapy or prevention of diseasesassociated with protein misfolding such as neurodegenerative diseasesand type 2 diabetes. Thus, there is a need in the art of a system whereone can identify therapeutic agents for diseases associated with proteinmisfolding which may have their therapeutic effect due to being eitherregulators of protein folding, and/or inhibitors of protein aggregation,and/or preventors and/or inhibitors of the process of fibrillogenesis,or those that can have an entirely different and possibly unknownmechanism of action. Furthermore, there is need that such a systemprovide a rapid and cost-effective screening method that will allow theidentification of agents useful in the treatment, prevention and cure ofdiseases associated with protein misfolding.

SUMMARY OF THE INVENTION

Diseases involving a misfolded protein have been identified in mammals(“misfolded protein diseases”). These diseases include Parkinson'sdisease; prion diseases (including Creutzfeldt-Jakob disease (CJD),Fatal Familia insomnia (FFI), Gerstmann-Straussler-Scheinker disease(GSS), mad cow disease, Scrapie, and kuru); Familial AmyloidPolyneuropathy, Tauopathies (including Pick Disease, Lobar Atrophy, andFrontotemporal dementia); Trinucleotide diseases (including Huntington'sdisease, spinocerebellar ataxia (SCA), dentatorubral pallidoluysianatrophy (DRPLA), Fragile-X syndrome, myotonic dystrophy, Haw RiverSyndrome, hereditary ataxias, Machado Joseph disease, and Kennedy'sdisease (spinobulbar muscular atrophy, SBMA)).

The present invention is based on the observation that proteins thatmisfold and are associated with a disease (“misfolded disease protein”)can be expressed in yeast as the basis for screening for therapeuticagents for the treatment of such a disease. Conditions and/or agentshave been identified that induce toxicity (“toxicity inducing agent”) ina yeast cell expressing a misfolded disease protein, such as huntingtinor alpha synuclein, which are associated with Huntington's disease andParkinson's disease, respectively. Furthermore, conditions and/or agentsthat induce toxicity in a yeast cell expressing a particular misfoldeddisease protein can be identified according to methods of the presentinvention. Identified conditions and/or agents can be implemented withyeast cells expressing the particular misfolded disease protein toidentify therapeutic agents that can be used for the disease associatedwith the misfolded disease protein. The screen uses viability of theyeast, which express a misfolded disease protein and in which toxicityis induced, to identify compounds that have therapeutic potential in thetreatment of the disease associated with the misfolded disease protein.An advantage of the screening methods is that an understanding of thephysiology and/or cell biology of the misfolded disease protein or ofthe etiology of a misfolded protein disease is not necessary to identifycandidate therapeutic compounds.

The present invention includes methods of screening for therapeuticagents for Huntington's disease. Such methods involve a yeast cell thatexpresses all or part of a huntingtin polypeptide, and which has or iscontacted with a condition or agent that induces toxicity in the yeastcell such that the yeast cell is no longer viable. Induction of toxicitywill lead to loss of viability in the yeast cell. Thus, viability of theyeast cell in the presence of a candidate compound indicates thecandidate compound is a candidate therapeutic agent. Viability is usedaccording to its ordinary meaning. It may be evaluated absolutely orrelatively, compared to controls. In some embodiments the yeast celldoes not express a wild-type Hsp-40 or a functional Hsp-40, which is acondition that induces toxicity in the yeast cell. As used herein,“contacting” a yeast cell with a compound refers to exposing,incubating, touching, associating, making accessible the yeast cell tothe compound.

In some embodiments, the huntingtin polypeptide comprises an N-terminalregion of a full-length huntingtin polypeptide. It is contemplated thatan N-terminal region of a huntingtin polypeptide can comprise theN-terminal region of exon 1 or all of exon 1, including a poly Q repeatregion. A poly Q repeat region refers to a region of a huntingtinpolypeptide that is characterized by a variable number of glutamineresidue repeats starting at position 18 of SEQ ID NO:4 (the HtQ103protein), SEQ ID NO:6 (the HtQ25 protein), and SEQ ID NO:9 (the Ht Exon1protein without poly Q repeats). In some embodiments of the invention,the poly Q region comprises 10, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100,110, 120, 130, 140, 150 or more glutamine residues; in specificembodiments, the poly Q region has 72 or 103 glutamine residues. Exon 1comprises amino acids 1-68 of the full length Huntingtin protein,however, this exon may comprise a variable number of glutamine residuesstarting at position 18 where the glutamine (CAG) repeats, in someembodiments, can be 25, 47, 72 or 103 glutamine residues long followedby the remaining 51 amino acids. The human gene sequence for the fulllength huntingtin polypeptide can be found within GenBank Accessionnumber NT_(—)006081, which is the sequence of chromosome 4, where thehuntingtin gene is located, incorporated herein by reference. In thepresent application, the number of glutamine residues in the poly Qregion, which is the region in exon 1 that is characterized by avariable number of glutamine residues, does not alter the amino acidpositions of residues downstream of the polyQ region. The term “HtQ25,”for example, refers to a huntingtin polypeptide that has a polyQ regionwith 25 glutamine residues, which is generally considered wild-type.

In some aspects of the screening methods, the yeast cell expresses apolypeptide that comprises a huntingtin polypeptide. The polypeptide mayalso comprise a non-huntingtin polypeptide. In some embodiments, thepolypeptide is a fusion protein comprising a huntingtin polypeptide andanother polypeptide, such as a reporter polypeptide. The reporterpolypeptide is any polypeptide that allows the polypeptide to bedetected or identified in a yeast cell. In some embodiments the reporterpolypeptide is a green fluorescent protein (GFP) or Sup35 (including theM and/or C region).

In some embodiments, a yeast cell expresses a mutated Hsp40 polypeptide,which may be exogenous or endogenous. The Hsp40 polypeptide may betruncated at either the C- or N-termini, or it may have an insertion,substitution, or internal deletion. In specific embodiments, the Hsp40polypeptide has a C-terminal deletion. The C-terminal deletion willinclude amino acid 352 of SEQ ID NO:8. It and other deletions maycomprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 3, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170,180, 190, 200, 210, 220, 230, 231, 240, 250 or more amino acidscontiguous with either amino acid 352 or amino acid 1, or any otheramino acid of SEQ ID NO:8. It is further contemplated that the Hsp40polypeptide may contain multiple mutations. An endogenous polypeptiderefers to a polypeptide expressed from a chromosomal (non-recombinant)nucleic acid molecule, whereas an exogenous polypeptide refers to oneexpressed outside the cell or expressed from a recombinant nucleic acidmolecule.

In further embodiments, a yeast cell, expressing or not expressingwild-type Hsp-40, is contacted with a toxicity inducing agent. The yeastmay be contacted with a candidate compound before, after, or duringcontacting with a toxicity inducing agent. A toxicity inducing agentincludes a carbon source, nitrogen source, salt, metal, liposome,antibiotic, anisomycin, bleomycin, caffeine, camptothecin,carbonyl-cyanide, daurorubicin, ethanol, formamide, GuHCL, or NEM, orother compounds identified in Table 3. With respect to a yeast cellexpressing a huntingtin polypeptide, in some embodiments, a toxicityinducing agent is a carbon source, such as arabinose or potassiumacetate, or a salt or metal, such as CdCl₂, CoCl₂, CsCl, FeCl₂, LiCl,NH₄Cl, RbCl, or ZnCl₂.

The claimed methods may also include comparing the viability of a yeastcell that was contacted with a candidate compound and that does notexpress a wild-type Hsp-40 with the viability of a yeast cell contactedwith the same candidate compound but that does express a wild-typeHsp40. Alternatively, the viability of a yeast cell that was contactedwith a candidate compound and that does not express a wild-type Hsp-40may be compared with the viability of a yeast cell that does not expressa wild-type Hsp-40 but not contacted with the candidate compound.Increased viability by the yeast cell contacted with the candidatecompound compared the yeast cell not contacted with the candidatecompound indicates that the candidate compound is a candidatetherapeutic agent. In other words, as with other embodiments of theinvention, absolute or relative viability (increased) in the presence ofthe candidate compound indicates the candidate compound is a candidatetherapeutic compound.

The present invention also concerns screening methods for therapeuticagents for Parkinson's disease involving yeast. In some embodiments ofthe invention a yeast cell expresses a polypeptide that includes all orpart of an alpha synuclein polypeptide, which is the misfolded diseaseprotein associated with Parkinson's disease. The yeast are contactedwith a toxicity inducing agent or a composition comprising a toxicityinducing agent. The yeast may be contacted with a candidate compoundbefore, after, or during contacting with a toxicity inducing agent.Absolute or relative viability in the presence of the candidate compoundindicates the candidate compound is a candidate therapeutic compound.

In some embodiments of the invention, the alpha synuclein polypeptide iswild-type (SEQ ID NO:2), while in other embodiments it is mutated. Themutation may be a deletion, insertion, or substitution in thepolypeptide. In specific aspects of the invention, the alpha synucleinpolypeptide comprises a A53T mutation, which is a substitution ofthreonine for alanine at position 53. In other aspects the alphasynuclein polypeptide comprises a A30P mutation, which is a substitutionof proline for alanine at position 53.

In still further embodiments, the alpha synuclein polypeptide iscomprised in a fusion protein, which may contain at least anotherpolypeptide. In some embodiments, the polypeptide is a fusion proteincomprising a huntingtin polypeptide and another polypeptide, such as areporter polypeptide. The reporter polypeptide is any polypeptide thatallows the polypeptide to be detected or identified in a yeast cell. Insome embodiments the reporter polypeptide is a green fluorescent protein(GFP) or Sup35 (including the M and/or C region).

A yeast expressing alpha synuclein in methods of the present inventionmay have a toxicity inducing condition or be contacted with a toxicityinducing agent. The toxicity inducing agent may be a carbon source,nitrogen source, salt, metal, azauracil, aurintrincarboxylic bleomycin,brefeldin A, camptothecin, chlorambucil, ethidium bromide, form amide,GuHCl, hydroxyurea, menadione, paraquat, or vanadate, or any othercompound listed in Table 3. In some embodiments, the carbon source isarabinose, ethanol, or glycerol, while in other embodiments, a nitrogensource is urea. In further embodiments, the toxicity inducing agent is asalt or metal, such as CaCl₂, CoCl₂, CsCl, or iron, magnesium, RbCl, orSrCl₂.

Generally speaking, all of the methods of the present invention mayinclude controls that involve comparing yeast cells in the present andabsence of candidate compounds, as well as yeast cells in the presenceand absence of toxicity inducing agents or toxicity inducing conditions.Such comparisons are discussed with respect to yeast expressing Hsp-40above, and may be employed with respect to any screen involving amisfolded disease protein. It is contemplated that any compositions ormethods discussed with respect to one embodiment may be employed in thecontext of other embodiments.

In some embodiments of the invention, viability is lost after 1 hour, 2hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 60hours, 72 hours, 84 hours, 96 hours, or more, but in less time than ifthe yeast cell had not been exposed to the condition or agent.

The candidate compounds of any of the methods of the invention may be asmall molecule or a nucleic acid. The candidate compounds may becomprised in a library or be processed for large-scale throughputscreening. The yeast that may be employed include Saccharomycescerevisiae or any other member of Saccharomycetales.

The present invention further encompasses methods of screening for atherapeutic agent for a protein misfolding disease comprising: a)contacting a yeast cell with a candidate compound, wherein the yeastcell expresses a polypeptide comprising a misfolded disease protein; b)contacting the yeast cell with a toxicity inducing agent; c) evaluatingthe yeast cell for viability, indicating the candidate compound is acandidate therapeutic agent. In some embodiments, the protein misfoldingdisease is Alzheimer's disease, Parkinson's disease, a Prion disease,Familial Amyloid Polyneuropathy, a Tauopathy, or a Trinucleotidedisease. It is specifically contemplated that the protein misfoldingdisease may a Trinucleotide disease, such as Huntington's disease. Inother embodiments the misfolded disease protein is huntingtin,62-amyloid, PrP, alpha synuclein, synphilin, transthyretin, Tau, ataxin1, ataxin 3, atrophin, or androgen receptor. It is also contemplatedthat the toxicity inducing agent may a carbon source, nitrogen source,salt, metal, chemotherapeutic agent, alcohol, translation inhibitor,NSAID, DNA intercalator, chelator, liposome, antibiotic, vitamin,proteasome inhibitor, anti-oxidant, or reducing agent. Furthermore, itis contemplated that instead of contacting the yeast cell with atoxicity inducing agent that the yeast may harbor a toxicity inducingcondition, such as a mutation in a chaperone protein. As discussedabove, embodiments discussed with respect to a screen for therapeuticagents for Huntington's or Parkinson's diseases may be employed withrespect to other misfolded protein diseases.

Other methods of the invention include methods of screening for atherapeutic agent for Huntington's disease comprising: a) contacting ayeast cell with a candidate compound, wherein the yeast cell expresses apolypeptide comprising a huntingtin polypeptide; b) incubating the yeastcell under conditions that allow for aggregation of the polypeptide; c)measuring the aggregation of the polypeptide; and comparing the level ofaggregation with the level of aggregation in a yeast cell not contactedwith the candidate compound. In some embodiments, the yeast cell has atoxicity inducing condition and/or is contacted with a toxicity inducingagent.

The invention also contemplates methods of screening for a therapeuticagent for Parkinson's disease comprising: a) contacting a yeast cellwith a candidate compound, wherein the yeast cell expresses apolypeptide comprising an alpha synuclein polypeptide; b) incubating theyeast cell under conditions that allow for aggregation of thepolypeptide; c) measuring the aggregation of the polypeptide; andcomparing the level of aggregation with the level of aggregation in ayeast cell not contacted with the candidate compound. In someembodiments, the yeast cell has a toxicity inducing condition and/or iscontacted with a toxicity inducing agent.

In addition to screening methods, compositions and methods for treatmentthat arise from the results of screening methods of the invention arealso included. Therapeutic agents for treating diseases and conditionsinvolving fibrillogenesis, including Parkinson's disease andHuntington's disease. In some embodiments of the invention, candidatecompounds that are screened may be employed in therapeutic methods andcompositions of the invention. In further embodiments, the candidatecompound is determined to be a candidate therapeutic agent based on itsperformance in screening assays. If cells incubated with the candidatecompound are more viable (based on characteristics that may include cellmorphology, number, growth rate, ability to be passaged, and/or abilityto be frozen and/or thawed) than cells not incubated with the candidatecompound, in some embodiments of the invention the candidate compound isa candidate therapeutic agent. The candidate therapeutic agent may beproduced or manufactured, or placed in a pharmaceutically acceptablecomposition. It is contemplated that any of the screening methodsdescribed herein may be employed with respect to thereapeutic methodsand compositions.

Methods of treating include administering to a patient in need oftreatment a therapeutic agent in an amount effective to achieve atherapeutic benefit. A “therapeutic benefit” in the context of thepresent invention refers to anything that promotes or enhances thewell-being of the subject with respect to the medical treatment of hiscondition, which includes treatment of fibrillogenesis diseases, such asHuntington's and Parkinson's diseases. A list of nonexhaustive examplesof this includes extension of the subject's life by any period of time,decrease or delay in the development of the disease, decrease in numberof plaques or fibrils, reduction in fibril growth, reduction in numberof misfolded proteins, delay in onset of lapse in mental capabilities,and a decrease in atrophy, or dementia to the subject that can beattributed to the subject's condition.

It is contemplated that compositions and steps discussed in the contextof an embodiment may be employed with respect to other embodimentsdiscussed herein.

As used herein “aggregation” is used to refer to a clustering oramassing of at least three separate polypeptides. Such “aggregation”precludes specific protein:protein interactions between polypeptides ofdifferent sequences, such as observed with yeast two hybrid assays.

As used herein the specification or claim(s) when used in conjunctionwith the word “comprising”, the words “a” or “an” may mean one or morethan one. As used herein “another” may mean at least a second or more.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Sis1p deletions/truncations.

FIG. 2. Expression of Ht fragments in yeast. Schematic representation ofHt-GFP fragments used in this study. Gray box, GFP; white boxes, aminoacids 1-68 of the N-terminal region of human Ht protein containing astretch of 25, 47, 72 or 103 glutamines (black box).

FIG. 3. Expression of alpha-synuclein fused to GFP under the control ofthe GAL1-10 promoter. Expression of WT and A53T is toxic to the cells.Similar phenotypes were observed with alpha-synuclein alone. Theseassays have been used in the screening methods to identify agents thatcan alleviate the observed toxicity.

FIG. 4. Schematic of the screen of a DNA library based on the disruptionof FRET between the proteins tagged with CFP and YFP.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Deposition of insoluble fibril proteins in tissues is a characteristicof diseases associated with protein misfolding. Most common of these areneurodegenerative diseases and diseases such as type 2 diabetes as well(see Table 1 for a list of diseases associated with protein misfolding).To date there is no known therapy or treatment that can dissolve theseprotein deposits. Therefore, agents that can prevention proteinaggregation and fibril formation are being actively sought. However,methods to identify potential candidate substances are lacking in theart.

The present inventors have developed a system which allows the rapididentification of candidate therapeutic agents that prevent and/orinhibit the process of protein aggregation leading to fibrillogenesisand protein deposition. The system is a yeast-based system, wherein ayeast cell is engineered to expresses a protein or polypeptide that isinvolved in fibril formation, for example, the yeast cell can express ahuntingtin polypeptide in the case of Huntington's disorder, orexpresses an alpha synuclein polypeptide in the case of Parkinson'sdisease, or express an amyloid protein in the case of a diseaseinvolving amyloidoses (also see Table 1 for a list of proteins that areassociated with fibril formation). In addition to this, in oneembodiment, the yeast cell also has a genetic background that causes theyeast cell to have reduced growth rates or no growth as a result ofexpressing the recombinant polypeptide in combination with the geneticbackground. In one example, the yeast cell has a mutant Hsp40 gene. Adecrease or inhibition of growth indicates toxicity of the recombinantfibril forming polypeptide in the yeast cell as a result of some changein expression or activity of other proteins or cellular factors thatinteract with the recombinant fibril polypeptide due to the change ingenetic background. This cytotoxic profile is correlated to human and/orother mammalian neurodegenerative state. Thus, if such a yeast cell isexposed to a candidate substance, one can screen for the potential ofthe agent to reverse cytotoxicity, which correlates to the ability ofthe agent to prevent cytotoxic and/or neurotoxic protein aggregation andfibril formation. TABLE 1 Disorders with Aberrant Protein DepositionCellular Localization of Disorder Protein aggregates Parkinson's Diseaseα-synuclein Cytoplasmic Alzheimer's Disease Amyloid-β ExtracellularAlzheimer's Disease Tau Intracellular Prion Diseases PrP ExtracellularHuntington's Disease huntingtin Variable intracellular Spinocerebellarataxia-1 Ataxin-1 Nuclear Spinocerebellar ataxia-2 Ataxin-2 NA*Spinocerebellar ataxia-3 Ataxin-3 Nuclear, perinuclear Spinocerebellarataxia-6 Calcium channel Cytoplasmic Spinocerebellar ataxia-7 Ataxin-7Nuclear Spinal and bulbar Androgen receptor Nuclear muscular atrophyDentatorubral Atrophin-1 Nuclear Pallidoluysian atrophy Amyotropiclateral SOD1 Cytoplasmic sclerosis Primary systemic Immunoglobulin NAamyloidosis light chain Famylial amyloid Transthyretin Extracellularpolyneuropathy Senile systemic Transthyretin Extracellular amyloidosisSecondary systemic Serum amyloid A NA amyloidosis Type 2 diabetes Isletamyloid NA polypeptide Injection-localized Insulin NA amyloidosisHemodialysis-related β2-microglobulin NA amyloidosis Hereditary cerebralCystatin-C NA amyloid angiopathy Finnish hereditary Gelsolin NA systemicamyloidosis Hereditary non- Lysozyme NA neuropathic*NA, not available

In an alternative embodiment, the yeast cell expressing the recombinantfibril forming protein or polypeptide, is exposed to a set of growthconditions that causes the yeast cell to have reduced or no growth. Forexample, one may contact the yeast cell with iron or a free radicalgenerator that causes oxidative stress to the cell. Again, a candidatesubstance can be contacted with this yeast cell to screen for potentialagents that can reverse yeast cytotoxicity, which is also correlated tothe ability of the agent to prevent cytotoxic protein aggregation andfibril formation.

Although, the mechanism of action of the agents so identified isirrelevant, some possible mechanisms include regulation of proteinfolding, inhibition of protein aggregation, solubilizing fibrils oraggregates, etc. The yeast-based screening systems of the presentinvention provide high-throughput and cost-effective screening methodsthat allow the identification of agents useful in the treatment,prevention and cure of diseases caused due to protein misfolding, and/oraggregation, and/or fibrillogenesis, including several neurodegenerativepathologies.

A. Yeast Cells

Yeast cells offer a powerful system to study the molecular basis ofdiseases associated with protein misfolding. It is well known thatgenetic and chemical screens can be easily performed in yeast as theorganism offers ease of manipulation. Yeast cells have been usedsuccessfully in the study several other disease-related human proteins,for example, CFTR and frataxin, which have corresponding homologues inyeast. Frataxin is a protein involved in a neurodegenerative disease.Therefore, yeast provides an ideal system to study proteins and genesthat are involved in human diseases due to the presence of correspondinghuman homologues. Diseases associated with amyloid and amyloid-likepropagation and specificity which constitute a major class of proteinmisfolding diseases can therefore be studied in yeast cells.Additionally, yeast cells have a non-mendelian inheritance factor,[PSI⁺], which propagates by a prion-like mechanism, a phenomenon thathas been extensively studied.

Any yeast strain may be used in context of the present invention. Someexamples of yeast cell strains that can be used in the present methodinclude Saccharomyces uvae, Saccharomyces kluyveri, Schizosaccharomycespombe, Saccharomyces uvarum, Kluyveromyces lactis, Hansenula polymorpha,Pichia pastoris, Pichia methanolica, Pichia kluyveri, Yarrowialipolytica, Candida sp., Candida utilis, Candida cacaoi, Geotrichum sp.and Geotrichum fermentans. The preferred yeast strain is Saccharomycescerevisiae.

As the invention concerns screening methods for a wide-variety ofpharmaceutical, chemical and genetic agents, one concern is that some ofthe candidate substances may not be either permeable into yeast cells,or may not be taken up by yeast cells, or may be rapidly metabolizedonce they enter into the yeast cell, or may be pumped out of the yeastcell. The present inventors contemplate using suitable mutations ofyeast strains designed to eliminate these problems. In one example, ayeast strain bearing mutations in 3 genes, the erg6, pdr1, and pdr3,which affect membrane efflux pumps and increasing permeability for drugsare contemplated of use. This particular strain have been usedsuccessfully in cancer research to identify growth regulators (seewebsite: http://dtp.nci.nih.gov for details).

B. Heat Shock Proteins

Heat-shock proteins (HSPs), which comprise several evolutionaryconserved protein families are induced in a physiological andbiochemical response to abrupt increases in temperature or exposure to avariety of other metabolic insults including heavy metals, oxidativestress, toxins, and amino acid analogs. This response occurs in allprokaryotic and eukaryotic cells and is characterized by repression ofnormal protein synthesis and initiation of transcription of HSP-encodinggenes. HSPs are a class of molecular chaperones and under normalconditions, constitutively expressed HSPs facilitate proper proteinfolding and maturation, promote protein translocation across membranes,and regulate hormone receptor and protein kinase activity. HSPs achievethis by associating with cellular proteins and regulating theirconformation.

All of the major HSPs, including those that are constitutively expressedand those that are expressed at very high levels in response to heat andother stresses, have related functions; they ameliorate problems causedby protein misfolding and aggregation. However, each major HSP familyhas a unique mechanism of action. Some promote the degradation ofmisfolded proteins (for example, Lon, Ubiquitin, and variousUbiquitin-conjugating enzymes); others bind to various types of foldingintermediates and prevent them from aggregating (for example, the HSP70sact by maintaining proteins in an unfolded conformation, whileHSP60/GroEL complexes act by facilitating protein folding), yet othershave a maturational or regulatory capacity on molecules includingsteroid hormone receptors (for example, the HSP90s), and yet another HSPpromotes the reactivation of proteins that have already aggregated(Hsp100) (Parsell and Lindquist, 1993; Parsell and Lindquist, 1994a andb).

Smaller HSPs can suppress aggregation and heat inactivation of variousproteins, including actin. Hsp40, the mammalian homolog of bacterialDnaJ heat shock protein, binds to new polypeptide chains as they arebeing synthesized on ribosomes and mediates their correct folding. Ithas been recently shown that, polyglutamine-expanded truncatedhuntingtin protein interacts with members of Hsp40 and Hsp70 families ofchaperones in a polyglutamine length-dependent manner (Krobitsch andLindquist, 2000; Jana et al., 2000).

Many Hsp40 proteins have been discovered in both prokaryotic andeukaryotic cells, with at least sixteen proteins in yeast and more than10 proteins in animals cells (see Table 2). These proteins have evolveddiverse cellular localizations and functions and have been divided intothree subgroups, depending upon the presence of certain conserved aminoacids in the J-domain and the presence of various other domains.Sequence alignment of the different yeast Hsp40 homologues to themammalian Hsp40 protein, HDJ-1, indicate that Sis1 is the mosthomologous with an amino acid identity of 40%. The yeast Sis1 and themammalian HDJ-1 are both members of class II. They contain an N-terminalJ-domain followed by a glycine-phenylalanine-rich region (GF-region) anda C-terminal region. Both HDJ-1 and Sis1 lack the zinc finger motifbetween the GF-region and the C-terminal domain. Both are heat-inducibleand found in the nucleus and the cytoplasm. TABLE 2 Heat Shock ProteinsClass 1 Class 2 Class3 Eubacteria DnaJ CbpA DjlA NolC Yeast Ydj1 Sis1,zuotin Sec63, Jem1 Mdj1 Caj1, Hlj1 Yjl162c, Ynl227c Scj1 Yir004w,Yjr097w Yfr041c Xdj1 Animals Hdj2 Hsj1a&bp 58ipk Tid56 Hdj1 Mtj1,auxilin Csp, Mida1

In the present invention, the model yeast system, Saccharomycescerevisiae, was used due the availability of multiple isogenic yeaststrains with different chaperone activities. However, as will berecognized by the skilled artisan, any other yeast strain may also beused. In one example of the present invention, wild-type yeast strainswere engineered to produce the N-terminal region of the huntingtin (Ht),protein with variable poly-glutamate (poly-Q) lengths, including 25, 47,72 or 103 residues which were fused to the green fluorescent protein(GFP). The production of N-terminal fragments of Ht, is a central eventin Huntington's disease (HD), which then leads to formation of Htaggregates in affected neurons during the natural progression of thedisease in both humans and in transgenic animal models. Expression ofthe Ht proteins was monitored by GFP fluorescence analysis using methodswell known in the art. Proteins with 25 glutamines (HtQ25) displayeddiffuse fluorescence, whereas proteins with longer glutamine tracts(HtQ47, HtQ72, or HtQ103) exhibited a proportionally greater tendency toaggregate. Differential sedimentation analysis of cell lysates revealedthat HtQ25 and HtQ47 were entirely soluble, whereas HtQ72 and HtQ103were mostly insoluble. These findings demonstrated that in yeast cells,as in mammalian cells, aggregation of Ht fragments depends upon thelength of the polyglutamine stretch.

The present inventors then investigated the effect of regulators ofprotein degradation on poly-glutamine-dependent aggregation. For this,strains with three different partial loss-of-function mutations in theproteasome/ubiquitination pathway (the ubiquitin-activating enzyme; thecatalytic subunit of the 20 S proteasome; or a subunit of the 19Sproteasome regulatory complex) were utilized and no difference in thefluorescence or sedimentation pattern was observed.

Changing the expression levels of most chaperones also did not affectaggregate formation. However, over-expressing Sis1 (the yeast homologueof mammalian Hdj-1), Hsp70 or Hsp104 modulated the aggregation of HtQ72and HtQ103. In Hsp104-deficient yeast cells, HtQ72 or HtQ103 remainedentirely soluble. Although no toxicity was observed with any of thesefragments, with or without aggregation, the inventors demonstrated thatthe aggregation of huntingtin in yeast cells depends on a balance ofchaperone activities in the cell.

As Sis1, which is the yeast homologue of Hsp40, is known to affect theaggregation state of huntingtin and is crucial for polyQ-inducedtoxicity in various model systems, the present inventors performed adetailed analysis of Sis1. Yeast strains engineered to express differentregions of the Sis1 protein were transformed with the huntingtin-GFPfusion constructs. Aggregation pattern of HtQ72 and HtQ103 were markedlyaltered by the production of mutant Sis1 proteins. Instead of a smallnumber of large aggregates a large number of smaller aggregates werepresent. Most notably, in one Sis1 construct the change in aggregationwas accompanied by a reduction in yeast cell viability. The inventorsfound that this Sis1-induced toxicity is reduced by co-expression ofHsp104. Hsp104 also reduces both aggregate formation and cell death in amammalian cell model of huntingtin toxicity and in a C. elegans modelemploying simple polyQ-GFP-fusions. Thus, the toxicity of huntingtininduced by Sis1 alterations in yeast correlate to Ht toxicity in humans.

Thus, the present inventors have developed systems that utilize yeast asa model system for the analysis of proteins that are involved in theformation of fibrils and/or proteins that aggregate to form insolubledeposits, exemplified by proteins such as huntingtin. The skilledartisan will recognize that huntingtin is merely a non-limiting example.The system and methods developed herein allow the identification ofagents that affect the conformational state of such proteins in a livingcell without the potential complications of toxicity (such as, theinduction of stress responses, the appearance of suppressor mutations,etc.).

To identify potential pharmaceutical and therapeutic agents that affectproteins that are involved in the formation of fibrils and/or proteinsthat aggregate to form insoluble deposits, the inventors contemplateexperiments that employ a yeast strain that expresses a truncated Sis1protein and a aggregating protein or fibril forming protein that causestoxicity, such as HtQ103. In the example of Ht proteins, the inventorscontemplate using yeast strains with the mutant Sis1 backgroundexpressing HtQ25 (control, not toxic), HtQ47 or HtQ72 (not toxic, butpotentially so), or HtQ103 (toxic). These yeast cells will be spotted inserial dilutions onto selective media with or without test agents.Increased growth rate on test plates compared to control plates willidentify compounds with a potential for reducing toxicity; a decreasedgrowth will identify compounds that might increase toxicity. Microscopicanalysis will determine whether these agents also affect aggregateformation and will use GFP-fusion proteins. This screen will also beperformed with yeast strains expressing only the N-terminal region ofHt, not fused to GFP. In addition, the inventors contemplate screening alibrary of FDA approved compounds for human use for identifyingtherapeutic agents for diseases involving aberrant protein deposition,and/or fibrillogenesis, and/or amyloidosis, and/or proteins aggregationto form insoluble deposits. The inventors also contemplate screening alarge scale combinatorial chemistry libraries and genomic and cDNAlibraries to identify chemical and genetic agents that can providetherapeutic benefit. Similar experiments are contemplated with otherfibril forming/aggregate forming proteins such as those listed in Table1.

Therefore, mutations in HSPs can result in diseases caused as a resultof protein misfolding, and protein aggregation among others. In thepresent invention, yeast cells with mutations in HSP genes have beenused to express recombinant proteins that are involved in diseasesassociated with protein misfolding. This results in yeast cells whichhave lower or no growth rates, indicating cytotoxic effects due tomisfolding of the recombinant protein in the cell that lacks the abilityto correct the misfolding. These yeast cells have been used to developscreening methods to identify agents that can correct protein foldingand thereby provide therapeutic or preventive benefits for diseasesinvolving protein misfolding.

C. Other Toxicity Inducing Agents

In other embodiments of the invention, some of the fibrilforming/aggregate forming proteins have been shown to have toxic effectswhen the yeast cell is subject to other toxicity inducing agents. Thetoxicity inducing agents can be a carbon source, nitrogen source, salt,metal, chemotherapeutic agent, alcohol, translation inhibitor, NSAID,DNA intercalator, chelator, liposome, antibiotic, vitamin, proteasomeinhibitor, anti-oxidant, or reducing agent (see some non-limitingexamples listed in Table 3). Thus, changes in growth conditions forexample, by exposure to one of the agents listed above, causes toxicityin yeast cells. The toxicity maybe due to oxidative stress or conditionsthat alter other stress response pathways of the yeast cell. Oxidativestress is defined here as any process that affect theoxidative/respiratory mechanism of a cell. This may be a result ofgeneration of free radicals or respiratory enzyme poisons. TABLE 3Putative Toxicity Inducing Agents Carbon Sources YPD Dextrose 2% (SD) pH4.9 Dextrose 2% (SD) pH 6.0 Dextrose 2% (SD) pH 6.8 FermentableGalactose 2% Maltose Melibiose Raffinose Sucrose Oleic acid Lauric acidArabinose 2% Non-Fermentable K-Acetate 3% Ethanol 3% Glycerol 2%Glycerol 20% Nitrogen Sources allantoin 1 mg ml−1 ammonia (NH4Cl) 1 mgml−1 glutamate 1 mg ml−1 glutamine 1 mg ml−1 ornithine 1 mg ml−1 proline1 mg ml−1 serine 1 mg ml−1 threonine 1 mg ml−1 Salts and Metals AlF3 1mM BaCl2 50 mM CaCl2 0.5 M CdCl2 20 μM CdCl2 50 μM CoCl2 750 μM CoCl2300 μM CsCl 0.1 M CsCl 25 mM CuSO₄ 0.5 mM CuSO₄ 2.5 mM CuSO₄ 5 mMFe₂(SO₄)₃ 8.5 mM Fe₂(SO₄)₄ 20 mM FeCl₂ 10 mM FeCl₂ 23 mM FeCl₂ 50 mMFeCl₃ 20 mM FeCl₃ 8.5 mM FeSO₄ 50 mM FeSO₄ 23 mM KI LiCl 0.3 M MgCl₂ 0.5M MgSO₄ 0.5 M MnCl₂ 4 mM NaCl 0.3 M NaCl 0.7 M NH₄Cl 0.9 M NiCl₂ 850 μMRbCl 0.2 M ZnCl₂ 2.5 mM ZnCl₂ 10 mM ZnCl₂ 5 mM Inhibitors1,10-phenanthroline 30 μg/ml 2,2-dipyridil 50 μg/ml 4-NQO 2.5 μg/ml4-NQO 2.5 μg.ml 5-azacytidine 100 μg/ml (toxic) 5-fluorocytosine 0.02mg/ml 5-fluorouracil 6-azauracil 30 μg/ml 8-hydroxyquinoline 26 μg/mlactinomycin D 45 μg/ml (no DMSO) actinomycin D DMSO anisomycin 20 μg/mlanisomycin 50 μg/ml antimycin A 1 μg/ml aspirin (Acetylsalicylic acid)aurintricarboxylic acid 100 μM BAPTA 20 mM benomyl 1 μg/ml (37° C.)benomyl 10 μg/ml (37° C.) benomyl 20 μg/ml (37° C.) benomyl 40 μg/mlbleomycin 10 μg/ml brefeldin A 100 μg/ml caffeine 1 mM caffeine 10 mMcalcofluor white (fluorescence B28) 1 mg/ml camptothecin 0.1 μg/mlcamptothecin 5 μg/ml canavanine 30 μM (SD-arg) carbonyl-cyanide m-chlorophenylhydrazone 1-3 μM cercosporamide 5 μg/ml cerulenin 0.5 μg/mlchlorambucil 3 mM) ciclopyroxolamine cinnarizine 100 μg/ml cycloheximide0.2 μg ml−1 (toxic & dangerous for environment) cycloheximide 3 μg ml−1daunomycin 0.05 mg/ml D-his 0.5 mM (L-pro) diamide 1 mM diamide 2 mMdiltiazem hydrochloride 2 mg/ml distamycin A SD 80-400 μMDL-C-allylglycine 0.025 mg/ml EDTA 1 mg/ml EGTA 10 mM emetine 2 ug/mlmg/ml erythromycin 200 μg/ml ethanol 10% ethanol 6% Ethidium bromide 25μg/ml Ethidium bromide 50 μg/ml Etoposide Fenpropinorph 0.3 μMFlufenamic acid Formamide 2% Formamide 3% griseofulvin 100 μg/ml GuHCl20 mM GuHCl 5 mM Hydroxyurea 10 mg/ml Hydroxyurea 5 mg/ml IbruprofenL-ethionine 1 ug/ml menadione 20 to 50 uM mevinolin 400 ug/mlmicocystin-LR 0.2 and 1 uM Na orthovanadate 3 mM nalidixic acid use 200ug/ml NBQX NEM 0.01 mM neomycin 5 mg/ml Nicotinic acid nocodazole 1μg/ml nocodazole 50 μg/ml nocodazole 10 μg/ml nystatin 2 μg/ml o-DNB 175μM oligomycin 1 μg/ml (YPGE) oligomycin 2.5 μg/ml (YPGE) olygomicin 5μg/ml (YPGE) papulacandin B 20 μg/ml paracetamol paraquat 1 mM (methylviologen) paraquat 10 mM paraquat 5 mM paromomycin 100 μg/ml paromomycin200 μg/ml paromomycin sulphate 2 mg/ml phenylethanol 2 mg/mlphenylethanol 5 mg/ml PMSF 4-5 mM protamine sulphate 750 μM protaminesulphate 250 μM quinolinic acid rapamycin 0.1 μg/ml SD-arg 1 ug/mlcanavanine SD-arg 30 ug/ml canavanine sodium fluoride 5 mM staurosporine0.1 μg/ml (37° C.) staurosporine 1 μg/ml (37° C.) streptonigrin 1 μg/mlThiamine Thiolutin 3 to 9 μg/ml trifluoperazine 20 uM tunicamycin 2.5μg/ml vanadate 1 mM vanadate 0.1 mM vanadate 2 mM vanadate 4 mM vanadate7 mM + KCl vanadate no KCl verapamil hydrochloride 100 μg/ml verrucarinA 2.45 μg/ml Liposomes DOSPA (lipofectamine) DOSPER DOGS (transfectam)DDAB DOPE Antibiotics Ampicillin Amphotericin B (Fungizone) 0.045 μg/mlAmphotericin B 45 μg/ml Chloramphenicol Cyclosporin A Kanamycin VitaminsVitamin A Vitamin B12 Vitamin C (ascorbate) Vitamin D Vitamin E(tocopherol) Vitamin K Proteasome Inhibitors ALLN 50 μM E64d 100 μM LLM50 μM MG132 50 μM quinacrine 2 μM chloroquine 4.2 μM chloroquine 10 μMclioquinol 5 μM (R)-(−)-3-hydroxybutirate*** D-beta-hydroxybutyrateDOPAMINE L-dihydroxyphenylalanine (L-DOPA) Amyloid related Congo Red 5μM Thiflavine S Thioflavine T chrysamine G 1.0 μM (3 to 30 μM in coscells) direct orange 6 μM direct yellow 20 0.5 μMN,N′-terephtalylidenebis- (4aminosalicylic acid) >100 μM4,4′-bis-(carboxyphenylamino)-3,3′- dimethoxybiphenyl >100 μMmyo-inositol 1.5 mg/ml epi-inositol 1.5 mg/ml scyllo-inostol 1.5 mg/mlDeoxycorticosterone Anti-oxidants 6-hydroxydopamine carvediloldeferoxamine mesylate Ferritin Estradiol Glutathione NO Reducing agents2-mercaptoethanol DTT Miscellaneous K-Acetate + PB UV Osmotic Stress KCl1.3 M Sorbitol 1.5 M Temperature ° C. 30 38 RT 14 ThermotoleranceEthanol Gradient Osmolytes Trehalose Glycerol 20% Control Plates forsolvents/additives 0.5 M KCl acetone 1% chloroform DMF DMSO 5% DMSO 1%DMSO/EtOH methanol 5%

D. Nucleic Acids

One embodiment of the present invention is to transfer nucleic acidsencoding a protein or polypeptide involved in protein aggregation and/orfibril formation, such as a misfolded disease protein, into a yeast cellso that the yeast cell expresses the protein. For example, one mayexpress alpha synuclein, huntingtin, transthyretin, β2-microglobulin, orany amyloid protein, such as beta-amyloid, alpha amyloid, islet amyloidpolypeptide, and the like (see Table 1 for other non-limiting examples).In one embodiment the nucleic acids encode a full-length, substantiallyfull-length, or functional equivalent form of such a protein orpolypeptide. In additional embodiments, a truncated polypeptide or apolypeptide with internal deletions is provided to a yeast cell. Inother embodiments the polypeptide is a human or other mammalianhomologue.

In other embodiments, the yeast cell may be also transfected withheat-shock protein. In yet other aspects the invention contemplatesco-transfecting the yeast cell with any protein that is involved ininteracting with other cellular proteins and assisting with proteinfolding, protein aggregation etc.

Thus, in some embodiments of the present invention, the development ofthe yeast-based screening system involves the transfection of a yeastcell with an expression construct encoding a protein or polypeptideinvolved in protein aggregation and/or fibril formation.

Certain aspects of the present invention concern at least one nucleicacid encoding a protein or polypeptide involved in protein aggregationand/or fibril formation molecule or a heat shock protein or a proteininvolved in interacting with other proteins. In certain aspects, thenucleic acid comprises a wild-type or mutant nucleic acid. In particularaspects, the nucleic acid encodes for at least one transcribed nucleicacid. In particular aspects, the nucleic acid encoding the protein orpolypeptide involved in protein aggregation and/or fibril formation, ora heat shock protein or a protein involved in interacting with otherproteins, encodes at least one protein, polypeptide, or peptide, orbiologically functional equivalent thereof. In other aspects, thenucleic acid encoding a protein or polypeptide involved in proteinaggregation and/or fibril formation encodes at least one nucleic acidsegment of SEQ ID NO:1 (alpha synuclein), SEQ ID NO:3 (HtQ103), SEQ IDNO:5 (HtQ25), SEQ ID NO:9 (Ht Exon 1 without any polyglutamine repeats)or at least one biologically functional equivalent thereof. In anotheraspect, the nucleic acid encoding a heat-shock protein encodes at leastone nucleic acid segment of SEQ ID NO:7 (HSP40 homologue of yeast, alsocalled Sis 1 in yeast cells) or at least one biologically functionalequivalent thereof.

The present invention also concerns the isolation or creation of atleast one recombinant construct or at least one recombinant host cellthrough the application of recombinant nucleic acid technology known tothose of skill in the art or as described herein. The recombinantconstruct or host cell may comprise at least one nucleic acid encoding aprotein or polypeptide involved in protein aggregation and/or fibrilformation, and may express at least one protein, polypeptide, orpeptide, involved in protein aggregation and/or fibril formation or atleast one biologically functional equivalent thereof.

In some embodiments the invention refers to DNA sequences identified byDatabase Accession numbers: Genbank NC_(—)001146, which is the accessionnumber for the chromosome on which the SIS1 gene is located, and SIS1 isreferenced by SGD ID S0004952; Genbank NM_(—)000345 for alpha-synuclein;and Genbank NT_(—)006081, for the accession number for chromosome 4where the Huntingtin gene is located.

As used herein “wild-type” refers to the naturally occurring sequence ofa nucleic acid at a genetic locus in the genome of an organism, andsequences transcribed or translated from such a nucleic acid. Thus, theterm “wild-type” also may refer to the amino acid sequence encoded bythe nucleic acid. As a genetic locus may have more than one sequence oralleles in a population of individuals, the term “wild-type” encompassesall such naturally occurring alleles. As used herein the term“polymorphic” means that variation exists (i.e., two or more allelesexist) at a genetic locus in the individuals of a population. As usedherein, “mutant” refers to a change in the sequence of a nucleic acid orits encoded protein, polypeptide, or peptide that is the result ofrecombinant DNA technology.

A nucleic acid may be made by any technique known to one of ordinaryskill in the art. Non-limiting examples of synthetic nucleic acid,particularly a synthetic oligonucleotide, include a nucleic acid made byin vitro chemical synthesis using phosphotriester, phosphite orphosphoramidite chemistry and solid phase techniques such as describedin EP 266,032, incorporated herein by reference, or via deoxynucleosideH-phosphonate intermediates as described by Froehler et al., 1986, andU.S. Pat. No. 5,705,629, each incorporated herein by reference. Anon-limiting example of enzymatically produced nucleic acid include oneproduced by enzymes in amplification reactions such as PCR™ (see forexample, U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,682,195, eachincorporated herein by reference), or the synthesis of oligonucleotidesdescribed in U.S. Pat. No. 5,645,897, incorporated herein by reference.A non-limiting example of a biologically produced nucleic acid includesrecombinant nucleic acid production in living cells, such as recombinantDNA vector production in bacteria (see for example, Sambrook et al.1989, incorporated herein by reference).

A nucleic acid may be purified on polyacrylamide gels, cesium chloridecentrifugation gradients, or by any other means known to one of ordinaryskill in the art (see for example, Sambrook et al. 1989, incorporatedherein by reference).

The term “nucleic acid” will generally refer to at least one molecule orstrand of DNA, RNA or a derivative or mimic thereof, comprising at leastone nucleobase, such as, for example, a naturally occurring purine orpyrimidine base found in DNA (e.g., adenine “A,” guanine “G,” thymine“T,” and cytosine “C”) or RNA (e.g. A, G, uracil “U,” and C). The term“nucleic acid” encompasses the terms “oligonucleotide” and“polynucleotide.” The term “oligonucleotide” refers to at least onemolecule of between about 3 and about 100 nucleobases in length. Theterm “polynucleotide” refers to at least one molecule of greater thanabout 100 nucleobases in length. These definitions generally refer to atleast one single-stranded molecule, but in specific embodiments willalso encompass at least one additional strand that is partially,substantially or fully complementary to the at least one single-strandedmolecule. Thus, a nucleic acid may encompass at least onedouble-stranded molecule or at least one triple-stranded molecule thatcomprises one or more complementary strand(s) or “complement(s)” of aparticular sequence comprising a strand of the molecule.

In certain embodiments, a “gene” refers to a nucleic acid that istranscribed. As used herein, a “gene segment” is a nucleic acid segmentof a gene. In certain aspects, the gene includes regulatory sequencesinvolved in transcription, or message production or composition. Inparticular embodiments, the gene comprises transcribed sequences thatencode for a protein, polypeptide or peptide. In keeping with theterminology described herein, an “isolated gene” may comprisetranscribed nucleic acid(s), regulatory sequences, coding sequences, orthe like, isolated substantially away from other such sequences, such asother naturally occurring genes, regulatory sequences, polypeptide orpeptide encoding sequences, etc. In this respect, the term “gene” isused for simplicity to refer to a nucleic acid comprising a nucleotidesequence that is transcribed, and the complement thereof. In particularaspects, the transcribed nucleotide sequence comprises at least onefunctional protein, polypeptide and/or peptide encoding unit. As will beunderstood by those in the art, this functional term “gene” includesboth genomic sequences, RNA or cDNA sequences, or smaller engineerednucleic acid segments, including nucleic acid segments of anon-transcribed part of a gene, including but not limited to thenon-transcribed promoter or enhancer regions of a gene. Smallerengineered gene nucleic acid segments may express, or may be adapted toexpress using nucleic acid manipulation technology, proteins,polypeptides, domains, peptides, fusion proteins, mutants and/or suchlike. Thus, a “truncated gene” refers to a nucleic acid sequence that ismissing a stretch of contiguous nucleic acid residues that encode aportion of a full-length protein or polypeptide involved in proteinaggregation and/or fibril formation. For example, a truncated gene maynot contain the nucleic acid sequence for the N-terminal region of theprotein or polypeptide involved in protein aggregation and/or fibrilformation or of a heat-shock protein gene.

“Isolated substantially away from other coding sequences” means that thegene of interest, in this case the gene encoding either a protein orpolypeptide involved in protein aggregation and/or fibril formation; ora heat-shock protein; or any molecular chaperone protein, forms thesignificant part of the coding region of the nucleic acid, or that thenucleic acid does not contain large portions of naturally-occurringcoding nucleic acids, such as large chromosomal fragments, otherfunctional genes, RNA or cDNA coding regions. Of course, this refers tothe nucleic acid as originally isolated, and does not exclude genes orcoding regions later added to the nucleic acid by recombinant nucleicacid technology.

In certain embodiments, the nucleic acid is a nucleic acid segment. Asused herein, the term “nucleic acid segment,” are smaller fragments of anucleic acid, such as for non-limiting example, those that encode onlypart of a peptide or polypeptide sequence involved in proteinaggregation and/or fibril formation. Thus, a “nucleic acid segment maycomprise any part of the gene sequence, of from about 2 nucleotides tothe full-length of the encoding region. In certain embodiments, the“nucleic acid segment” encompasses the full-length gene sequence.

Various nucleic acid segments may be designed based on a particularnucleic acid sequence, and may be of any length. By assigning numericvalues to a sequence, for example, the first residue is 1, the secondresidue is 2, etc., an algorithm defining all nucleic acid segments canbe created:n to n+y

where n is an integer from 1 to the last number of the sequence and y isthe length of the nucleic acid segment minus one, where n+y does notexceed the last number of the sequence. Thus, for a 10-mer, the nucleicacid segments correspond to bases 1 to 10, 2 to 11, 3 to 12 . . . and/orso on. For a 15-mer, the nucleic acid segments correspond to bases 1 to15, 2 to 16, 3 to 17 . . . and/or so on. For a 20-mer, the nucleicsegments correspond to bases 1 to 20, 2 to 21, 3 to 22 . . . and/or soon. In certain embodiments, the nucleic acid segment may be a probe orprimer.

The nucleic acid(s) of the present invention, which encode either aprotein or polypeptide involved in protein aggregation and/or fibrilformation; or a heat-shock protein; or any molecular chaperone protein,regardless of the length of the sequence itself, may be combined withother nucleic acid sequences, including but not limited to, promoters,enhancers, polyadenylation signals, restriction enzyme sites, multiplecloning sites, coding segments, and the like, to create one or morenucleic acid construct(s). The overall length may vary considerablybetween nucleic acid constructs. Thus, a nucleic acid segment of almostany length may be employed, with the total length preferably beinglimited by the ease of preparation or use in the intended recombinantnucleic acid protocol.

(a) Nucleic Acid Vectors for the Expression of Screening MethodComponents in Yeast Cells

A gene encoding a component of the assay system of the invention, suchas misfolded disease protein; or a heat shock protein; or any othermolecular chaperone; or even a candidate substance that has therapeuticvalue for protein misfolding diseases may be transfected into a yeastcell using a nucleic acid vector, including but are not limited to,plasmids, linear nucleic acid molecules, artificial chromosomes andepisomal vectors. Yeast plasmids are naturally preferred and threesystems used for recombinant plasmid expression and replication in yeastinclude:

1. Integrating plasmids: An example of such a plasmid is YIp, which ismaintained at one copy per haploid genome, and is inherited in Mendelianfashion. Such a plasmid, containing a gene of interest, a bacterialorigin of replication and a selectable gene (typically anantibiotic-resistance marker), is produced in bacteria. The purifiedvector is linearized within the selectable gene and used to transformcompetent yeast cells. Regardless of the type of plasmid used, yeastcells are typically transformed by chemical methods (e.g. as describedby Rose et al., 1990, Methods in Yeast Genetics, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.). The cells are typicallytreated with lithium acetate to achieve transformation efficiencies ofapproximately 10⁴ colony-forming units (transformed cells)/μg of DNA.Yeast perform homologous recombination such that the cut, selectablemarker recombines with the mutated (usually a point mutation or a smalldeletion) host gene to restore function. Transformed cells are thenisolated on selective media.

2. Low copy-number ARS-CEN: One example is YCp and such plasmids containthe autonomous replicating sequence (ARS 1), a sequence of approximately700 bp which, when carried on a plasmid, permits its replication inyeast, and a centromeric sequence (CEN4), the latter of which allowsmitotic stability. These are usually present at 1-2 copies per cell.Removal of the CEN sequence yields a YRp plasmid, which is typicallypresent in 100-200 copes per cell; however, this plasmid is bothmitotically and meiotically unstable.

3. High-copy-number 2p circles: These plasmids contain a sequenceapproximately 1 kb in length, the 2μ sequence, which acts as a yeastreplicon giving rise to higher plasmid copy number; however, theseplasmids are unstable and require selection for maintenance. Copy numberis increased by having on the plasmid a selection gene operativelylinked to a crippled promoter. This is usually the LEU2 gene with atruncated promoter (LEU2-d), such that low levels of the Leu2p proteinare produced; therefore, selection on a leucine-depleted medium forcesan increase in copy number in order to make an amount of Leu2psufficient for cell growth.

Examples of yeast plasmids useful in the invention include the YRpplasmids (based on autonomously-replicating sequences, or ARS) and theYEp plasmids (based on the 2μ circle), of which examples are YEp24 andthe YEplac series of plasmids (Gietz and Sugino, 1988). (See Sikorski,“Extrachromsomoal cloning vectors of Saccharomyces cerevisiae”, inPlasmid, A Practical Approach, Ed. K. G. Hardy, IRL Press, 1993; andYeast Cloning Vectors and Genes, Current Protocols in Molecular Biology,Section II, Unit 13.4, Eds., Ausubel et al., 1994).

In addition to a yeast origin of replication, yeast plasmid sequencestypically comprise an antibiotic resistance gene, a bacterial origin ofreplication (for propagation in bacterial cells) and a yeast nutritionalgene for maintenance in yeast cells. The nutritional gene (or“auxotrophic marker”) is most often one of the following: TRP1Phosphoribosylanthranilate isomerase, which is a component of thetryptophan biosynthetic pathway); URA43 (Orotidine-5′-phosphatedecarboxylase, which takes part in the uracil biosynthetic pathway);LEU2 (3-Isopropylmalate dehydrogenase, which is involved with theleucine biosynthetic pathway); HIS3 (Imidazoleglycerolphosphatedehydratase, or IGP dehydratase); or LYS2 (α-aminoadipate-semialdehydedehydrogenase, part of the lysine biosynthetic pathway.

(b) Promoters and Enhancers

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.It may contain genetic elements at which regulatory proteins andmolecules may bind, such as RNA polymerase and other transcriptionfactors, to initiate the specific transcription a nucleic acid sequence.The phrases “operatively positioned,” “operatively linked,” “undercontrol,” and “under transcriptional control” mean that a promoter is ina correct functional location and/or orientation in relation to anucleic acid sequence to control transcriptional initiation and/orexpression of that sequence.

A promoter generally comprises a sequence that functions to position thestart site for RNA synthesis. The best known example of this is the TATAbox, but in some promoters lacking a TATA box, such as, for example, thepromoter for the mammalian terminal deoxynucleotidyl transferase geneand the promoter for the SV40 late genes, a discrete element overlyingthe start site itself helps to fix the place of initiation. Additionalpromoter elements regulate the frequency of transcriptional initiation.Typically, these are located in the region 30-110 bp upstream of thestart site, although a number of promoters have been shown to containfunctional elements downstream of the start site as well. To bring acoding sequence “under the control of” a promoter, one positions the 5′end of the transcription initiation site of the transcriptional readingframe “downstream” of (i.e., 3′ of) the chosen promoter. The “upstream”promoter stimulates transcription of the DNA and promotes expression ofthe encoded RNA.

The spacing between promoter elements frequently is flexible, so thatpromoter function is preserved when elements are inverted or movedrelative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either cooperatively or independently to activatetranscription. A promoter may or may not be used in conjunction with an“enhancer,” which refers to a cis-acting regulatory sequence involved inthe transcriptional activation of a nucleic acid sequence.

A promoter may be one naturally associated with a nucleic acid sequence,as may be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant or heterologous promoter, which refers to a promoterthat is not normally associated with a nucleic acid sequence in itsnatural environment. A recombinant or heterologous enhancer refers alsoto an enhancer not normally associated with a nucleic acid sequence inits natural environment. Such promoters or enhancers may includepromoters or enhancers of other genes, and promoters or enhancersisolated from any other virus, or prokaryotic or eukaryotic cell, andpromoters or enhancers not “naturally occurring,” i.e., containingdifferent elements of different transcriptional regulatory regions,and/or mutations that alter expression. For example, promoters that aremost commonly used in recombinant DNA construction include theβ-lactamase (penicillinase), lactose and tryptophan (trp) promotersystems. In addition to producing nucleic acid sequences of promotersand enhancers synthetically, sequences may be produced using recombinantcloning and/or nucleic acid amplification technology, including PCR™, inconnection with the compositions disclosed herein (see U.S. Pat. Nos.4,683,202 and 5,928,906, each incorporated herein by reference).Furthermore, it is contemplated the control sequences that directtranscription and/or expression of sequences within non-nuclearorganelles such as mitochondria, chloroplasts, and the like, can beemployed as well. Control sequences comprising promoters, enhancers andother locus or transcription controlling/modulating elements are alsoreferred to as “transcriptional cassettes”.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in theorganelle, cell type, tissue, organ, or organism chosen for expression.Those of skill in the art of molecular biology generally know the use ofpromoters, enhancers, and cell type combinations for protein expression,(see, for example Sambrook et al., 1989, incorporated herein byreference). The promoters employed may be constitutive, tissue-specific,inducible, and/or useful under the appropriate conditions to direct highlevel expression of the introduced DNA segment, such as is advantageousfor gene therapy or for applications such as the large-scale productionof recombinant proteins and/or peptides. The promoter may beheterologous or endogenous.

Use of a T3, T7 or SP6 cytoplasmic expression system is another possibleembodiment. Eukaryotic cells can support cytoplasmic transcription fromcertain bacterial promoters if the appropriate bacterial polymerase isprovided, either as part of the delivery complex or as an additionalgenetic expression construct.

Various inducible elements/promoters/enhancers that may be employed, inthe context of the present invention, to regulate the expression of aRNA. Inducible elements are regions of a nucleic acid sequence that canbe activated in response to a specific stimulus. Some examples of yeastspecific promoters include inducible promoters such as Gal1-10, Gal1,GalL, GalS, repressible promoter Met25, and constitutive promoters suchas glyceraldehyde 3-phosphate dehydrogenase promoter (GPD), alcoholdehydrogenase promoter (ADH), translation-elongation factor-1-alphapromoter (TEF), cytochrome c-oxidase promoter (CYC1), MRP7 etc.Autonomously replicating expression vectors of yeast containingpromoters inducible by glucocorticoid hormones have also been described(Picard et al., 1990), these include the glucorticoid responsive element(GRE). These and other examples are described in Mumber et al., 1995;Ronicke et al., 1997; Gao, 2000, all incorporated herein by reference.Yet other yeast vectors containing constitutive or inducible promoterssuch as alpha factor, alcohol oxidase, and PGH may be used. For reviews,see Ausubel et al. and Grant et al., 1987. Additionally anypromoter/enhancer combination (as per the Eukaryotic Promoter Data BaseEPDB) could also be used to drive expression of genes.

The identity of tissue-specific promoters or elements, as well as assaysto characterize their activity, is well known to those of skill in theart. Non-limiting examples of such regions include the human LIMK2 gene(Nomoto et al., 1999), the somatostatin receptor 2 gene (Kraus et al.,1998), murine epididymal retinoic acid-binding gene (Lareyre et al.,1999), human CD4 (Zhao-Emonet et al., 1998), mouse alpha2 (XI) collagen(Tsumaki, et al., 1998), D1A dopamine receptor gene (Lee, et al., 1997),insulin-like growth factor II (Wu et al., 1997), and human plateletendothelial cell adhesion molecule-1 (Almendro et al., 1996).

Typically promoters and enhancers that control the transcription ofprotein encoding genes in eukaryotic cells are composed of multiplegenetic elements. The cellular machinery is able to gather and integratethe regulatory information conveyed by each element, allowing differentgenes to evolve distinct, often complex patterns of transcriptionalregulation.

Enhancers were originally detected as genetic elements that increasedtranscription from a promoter located at a distant position on the samemolecule of DNA. This ability to act over a large distance had littleprecedent in classic studies of prokaryotic transcriptional regulation.Subsequent work showed that regions of DNA with enhancer activity areorganized much like promoters. That is, they are composed of manyindividual elements, each of which binds to one or more transcriptionalproteins.

The basic distinction between enhancers and promoters is operational. Anenhancer region as a whole must be able to stimulate transcription at adistance; this need not be true of a promoter region or its componentelements. On the other hand, a promoter must have one or more elementsthat direct initiation of RNA synthesis at a particular site and in aparticular orientation, whereas enhancers lack these specificities.Aside from this operational distinction, enhancers and promoters arevery similar entities.

Promoters and enhancers have the same general function of activatingtranscription in the cell. They are often overlapping and contiguous,often seeming to have a very similar modular organization. Takentogether, these considerations suggest that enhancers and promoters arehomologous entities and that the transcriptional activator proteinsbound to these sequences may interact with the cellular transcriptionalmachinery in fundamentally the same way.

A signal that may prove useful is a polyadenylation signal (hGH, BGH,SV40). The use of internal ribosome binding sites (IRES) elements areused to create multigene, or polycistronic, messages. IRES elements areable to bypass the ribosome scanning model of 5′-methylatedcap-dependent translation and begin translation at internal sites(Pelletier and Sonenberg, 1988). IRES elements from two members of thepicornavirus family (polio and encephalomyocarditis) have been described(Pelletier and Sonenberg, 1988), as well as an IRES from a mammalianmessage (Macejak and Sarnow, 1991). IRES elements can be linked toheterologous open reading frames. Multiple open reading frames can betranscribed together, each separated by an IRES, creating polycistronicmessages. By virtue of the IRES element, each open reading frame isaccessible to ribosomes for efficient translation. Multiple genes can beefficiently expressed using a single promoter/enhancer to transcribe asingle message.

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

(c) Multiple Cloning Sites

Vectors used to transform the yeast cells in the present invention caninclude a multiple cloning site (MCS), which is a nucleic acid regionthat contains multiple restriction enzyme sites, any of which can beused in conjunction with standard recombinant technology to digest thevector (see, for example, Carbonelli et al., 1999, Levenson et al.,1998, and Cocea, 1997, incorporated herein by reference.) “Restrictionenzyme digestion” refers to catalytic cleavage of a nucleic acidmolecule with an enzyme that functions only at specific locations in anucleic acid molecule. Many of these restriction enzymes arecommercially available. Use of such enzymes is widely understood bythose of skill in the art. Frequently, a vector is linearized orfragmented using a restriction enzyme that cuts within the MCS to enableexogenous sequences to be ligated to the vector. “Ligation” refers tothe process of forming phosphodiester bonds between two nucleic acidfragments, which may or may not be contiguous with each other.Techniques involving restriction enzymes and ligation reactions are wellknown to those of skill in the art of recombinant technology.

(d) Splicing Sites

Most transcribed eukaryotic RNA molecules will undergo RNA splicing toremove introns from the primary transcripts. Vectors containing genomiceukaryotic sequences may require donor and/or acceptor splicing sites toensure proper processing of the transcript for protein expression (see,for example, Chandler et al., 1997, herein incorporated by reference.)

(e) Termination Signals

The vectors or constructs of the present invention will generallycomprise at least one termination signal. A “termination signal” or“terminator” is comprised of the DNA sequences involved in specifictermination of an RNA transcript by an RNA polymerase. Thus, in certainembodiments a termination signal that ends the production of an RNAtranscript is contemplated. A terminator may be necessary in vivo toachieve desirable message levels.

In eukaryotic systems, the terminator region may also comprise specificDNA sequences that permit site-specific cleavage of the new transcriptso as to expose a polyadenylation site. This signals a specializedendogenous polymerase to add a stretch of about 200 A residues (polyA)to the 3′ end of the transcript. RNA molecules modified with this polyAtail appear to more stable and are translated more efficiently. Thus, inother embodiments involving eukaryotes, it is preferred that thatterminator comprises a signal for the cleavage of the RNA, and it ismore preferred that the terminator signal promotes polyadenylation ofthe message. The terminator and/or polyadenylation site elements canserve to enhance message levels and to minimize read through from thecassette into other sequences.

Terminators contemplated for use in the invention include any knownterminator of transcription described herein or known to one of ordinaryskill in the art, including but not limited to, for example, thetermination sequences of genes, such as for example the bovine growthhormone terminator or viral termination sequences, such as for examplethe SV40 terminator. In certain embodiments, the termination signal maybe a lack of transcribable or translatable sequence, such as due to asequence truncation.

(f) Polyadenylation Signals

In eukaryotic gene expression, one will typically include apolyadenylation signal to effect proper polyadenylation of thetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed. Some examples include the SV40 polyadenylationsignal or the bovine growth hormone polyadenylation signal, convenientand known to function well in various target cells. Polyadenylation mayincrease the stability of the transcript or may facilitate cytoplasmictransport.

(g) Origins of Replication

In order to propagate a vector of the invention in a host cell, it maycontain one or more origins of replication sites (often termed “ori”),which is a specific nucleic acid sequence at which replication isinitiated. Alternatively, an autonomously replicating sequence (ARS) canbe employed if the host cell is yeast.

(h) Selectable and Screenable Markers

In certain embodiments of the invention, yeast cells transduced with theconstructs of the present invention may be identified in vitro or invivo by including a marker in the expression vector. Such markers wouldconfer an identifiable change to the transduced cell permitting easyidentification of cells containing the expression vector. Generally, aselectable marker is one that confers a property that allows forselection. A positive selectable marker is one in which the presence ofthe marker allows for its selection, while a negative selectable markeris one in which its presence prevents its selection. An example of apositive selectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genetic constructs thatconfer resistance to kanamycin, neomycin, puromycin, hygromycin, DHFR,GPT, zeocin and histidinol are useful selectable markers. In addition tomarkers conferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis isfluorescence analysis, are also contemplated. Other reporterpolypeptides used in the present invention include Sup35p or other yeastprions. Additionally, auxotrophic markers such as leu, ura, trp, his,and the like for selection on different media. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be utilized. One of skill inthe art would also know how to employ immunologic markers, possibly inconjunction with FACS analysis. The marker used is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable and screenable markers are well known to one of skill in theart.

(i) Oligonucleotide Probes and Primers

Naturally, the present invention also encompasses DNA segments that arecomplementary, or essentially complementary, to the sequences encodingproteins or polypeptides involved in protein aggregation and/or fibrilformation or heat-shock proteins, for example those set forth in SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7. Nucleic acid sequencesthat are “complementary” are those that are capable of base-pairingaccording to the standard Watson-Crick complementary rules. As usedherein, the term “complementary sequences” means nucleic acid sequencesthat are substantially complementary, as may be assessed by the samenucleotide comparison set forth above, or as defined as being capable ofhybridizing to the nucleic acid segment encoding proteins orpolypeptides involved in protein aggregation and/or fibril formation orto heat-shock proteins, under relatively stringent conditions such asthose described herein. Such sequences may encode the entire proteininvolved in protein aggregation and/or fibril formation, or heat-shockproteins, or may be a fragment thereof.

The nucleic acid detection techniques and conditions described hereinserve both to define the functionally equivalent nucleic acids of theinvention, as outlined structurally above, and to describe certainmethods by which the yeast cells transformed with proteins orpolypeptides involved in protein aggregation and/or fibril formationsequences, or heat-shock protein sequences, may be screened, selected,and characterized.

Hybridizing fragments should be of sufficient length to provide specifichybridization to a RNA or DNA tissue sample. The use of a hybridizationprobe of between about 10-14 or 15-20 and about 100 nucleotides inlength allows the formation of a duplex molecule that is both stable andselective. Molecules having complementary sequences over stretchesgreater than 20 bases in length are generally preferred, in order toincrease stability and selectivity of the hybrid, and thereby improvethe quality and degree of particular hybrid molecules obtained.

Sequences of 17 bases long should occur only once in the human genomeand, therefore, suffice to specify a unique target sequence. Althoughshorter oligomers are easier to make and increase in vivo accessibility,numerous other factors are involved in determining the specificity ofhybridization. Both binding affinity and sequence specificity of anoligonucleotide to its complementary target increases with increasinglength. It is contemplated that exemplary oligonucleotides of 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100 or more base pairs will be used,although others are contemplated. Longer polynucleotides encoding 250,300, 500, 600, 700, 800, 900, 1000, 1100, 1200 and longer arecontemplated as well. Such oligonucleotides will find use, for example,as probes in Southern and Northern blots and as primers in amplificationreactions.

Accordingly, the nucleotide sequences of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of genes or RNAs or to provide primers for amplification ofDNA or RNA from tissues. Depending on the application envisioned, onewill desire to employ varying conditions of hybridization to achievevarying degrees of selectivity of probe towards target sequence.

For applications requiring high selectivity, one will typically desireto employ relatively stringent conditions to form the hybrids, e.g., onewill select relatively low salt and/or high temperature conditions, suchas provided by about 0.02 M to about 0.10 M NaCl at temperatures ofabout 50° C. to about 70° C. Such high stringency conditions toleratelittle, if any, mismatch between the probe and the template or targetstrand, and would be particularly suitable for isolating specific genesor detecting specific mRNA transcripts. It is generally appreciated thatconditions can be rendered more stringent by the addition of increasingamounts of formamide.

For certain applications, for example, substitution of amino acids bysite-directed mutagenesis, it is appreciated that lower stringencyconditions are required. Under these conditions, hybridization may occureven though the sequences of probe and target strand are not perfectlycomplementary, but are mismatched at one or more positions. Conditionsmay be rendered less stringent by increasing salt concentration anddecreasing temperature. For example, a medium stringency condition couldbe provided by about 0.1 to 0.25 M NaCl at temperatures of about 37° C.to about 55° C., while a low stringency condition could be provided byabout 0.15 M to about 0.9 M salt, at temperatures ranging from about 20°C. to about 55° C. Thus, hybridization conditions can be readilymanipulated, and thus will generally be a method of choice depending onthe desired results.

In other embodiments, hybridization may be achieved under conditions of,for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 10 mMdithiothreitol, at temperatures between approximately 20° C. to about37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 μM MgCl₂, attemperatures ranging from approximately 40° C. to about 72° C.

One method of using probes and primers of the present invention is inthe search for genes related to misfolded disease proteins or, moreparticularly, homologs of misfolded disease proteins from other species.Normally, the target DNA will be a genomic or cDNA library, althoughscreening may involve analysis of RNA molecules. By varying thestringency of hybridization, and the region of the probe, differentdegrees of homology may be discovered.

Another way of exploiting probes and primers of the present invention isin site-directed, or site-specific mutagenesis. Site-specificmutagenesis is a technique useful in the preparation of individualpeptides, or biologically functional equivalent proteins or peptides,through specific mutagenesis of the underlying DNA. The techniquefurther provides a ready ability to prepare and test sequence variants,incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

The technique typically employs a bacteriophage vector that exists inboth a single stranded and double stranded form. Typical vectors usefulin site-directed mutagenesis include vectors such as the M13 phage.These phage vectors are commercially available and their use isgenerally well known to those skilled in the art. Double strandedplasmids are also routinely employed in site directed mutagenesis, whicheliminates the step of transferring the gene of interest from a phage toa plasmid.

In general, site-directed mutagenesis is performed by first obtaining asingle-stranded vector, or melting of two strands of a double strandedvector which includes within its sequence a DNA sequence encoding thedesired protein. An oligonucleotide primer bearing the desired mutatedsequence is synthetically prepared. This primer is then annealed withthe single-stranded DNA preparation, taking into account the degree ofmismatch when selecting hybridization conditions, and subjected to DNApolymerizing enzymes such as E. coli polymerase I Klenow fragment, inorder to complete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed wherein one strand encodes the originalnon-mutated sequence and the second strand bears the desired mutation.This heteroduplex vector is then used to transform appropriate cells,such as E. coli cells, and clones are selected that include recombinantvectors bearing the mutated sequence arrangement.

The preparation of sequence variants of the selected gene usingsite-directed mutagenesis is provided as a means of producingpotentially useful species and is not meant to be limiting, as there areother ways in which sequence variants of genes may be obtained. Forexample, recombinant vectors encoding the desired gene may be treatedwith mutagenic agents, such as hydroxylamine, to obtain sequencevariants.

In certain embodiments, it will be advantageous to employ nucleic acidsequences of the present invention in combination with an appropriatemeans, such as a label, for determining hybridization. A wide variety ofappropriate indicator means are known in the art, including fluorescent,radioactive, enzymatic or other ligands, such as avidin/biotin, whichare capable of being detected.

In certain embodiments, one may desire to employ a fluorescent label,electroluminescence or an enzyme tag such as urease, alkalinephosphatase or peroxidase, instead of radioactive or otherenvironmentally undesirable reagents. In the case of enzyme tags,calorimetric indicator substrates are known which can be employed toprovide a detection means visible to the human eye orspectrophotometrically, to identify specific hybridization withcomplementary nucleic acid-containing samples.

In general, it is envisioned that the hybridization probes describedherein will be useful both as reagents in solution hybridization, as inPCR™, for detection of expression of corresponding genes, as well as inembodiments employing a solid phase. In embodiments involving a solidphase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to hybridization with selected probes under desiredconditions.

The selected conditions will depend on the particular circumstancesbased on the particular criteria required (depending, for example, onthe G+C content, type of target nucleic acid, source of nucleic acid,size of hybridization probe, etc.). Following washing of the hybridizedsurface to remove non-specifically bound probe molecules, hybridizationis detected, or even quantified, by means of the label.

E. Protein, Polypeptides, and Peptides

The invention contemplates the use of a polypeptide or a proteinsencoding a misfolded disease protein or a heat shock protein. In someembodiments a full-length or a substantially full-length misfoldeddisease protein/polypeptide or heat shock protein may be used. The term“full-length” refers to a misfolded disease polypeptide or heat shockprotein that contains at least all the amino acids encoded by themisfolded disease protein cDNA or heat shock protein cDNA. The term“substantially full-length” in the context of a misfolded diseaseprotein refers to a misfolded disease protein/polypeptide that containsat least 80% of the contiguous amino acids of the full-length misfoldeddisease protein/polypeptide. However, it is also contemplated that amisfolded disease protein/polypeptides or heat shock protein containingat least about 85%, 90%, and 95% of SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, or SEQ ID NO:9 are within the scope of the inventionas a “substantially full-length” misfolded disease protein/polypeptide.

In various embodiments different lengths of the proteins/polypeptides ofthe present invention may be used. For example, only functionally activedomains of the proteins may be used. Thus, a protein/polypeptide segmentof almost any length may be employed.

In a non-limiting example, one or more proteins or polypeptides may beprepared that include a contiguous stretch of amino acids identical toor complementary to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,or SEQ ID NO:9. Such a stretch of amino acids, may be about 3, about 4,about 5, about 6, about 7, about 8, about 9, about 10, about 15, about20, about 25, about 30, about 35, about 40, about 45, about 50, about55, about 60, about 65, about 70, about 75, about 80, about 85, about90, about 95, about 100, about 105, about 110, about 115, about 120,about 125, about 130, about 135, about 140, about 145, about 150, about155, about 160, about 165, about 170, about 175, about 180, about 185,about 190, about 195, about 200, about 210, about 220, about 230, about240, about 250, about 260, about 270, about 280, about 290, about 300,about 310, about 320, about 330, about 340, about 350, about 360, about370, about 380, about 390, about 400, about 410, about 420, about 430,about 440, about 450, about 460, about 470, about 480, about 490, about500, about 510, about 520, about 530, about 540, about 550, about 560,about 570, about 580, about 590, about 600, about 610, about 620, about630, about 640, about 650, about 660, about 670, about 680, about 690,to about 700 amino acids in length or longer, including all intermediatelengths and intermediate ranges. It will be readily understood that“intermediate lengths” and “intermediate ranges,” as used herein, meansany length or range including or between the given values (i.e., allintegers including and between such values).

It is also contemplated that in the case of polyglutamine (pQ)containing polypeptide sequences the numeric order of the amino acidswill not be changed by the number of the pQ repeats. In the example of ahuntingtin's polypeptide encoded by Exon 1, the polypeptide is comprisedof 68 amino acids, excluding pQ repeats. The pQ repeats typically beginat position 18. SEQ ID NO:9 is an example where there are no pQ repeats.However, in other examples variable number of pQ repeats are present,for example, SEQ ID NO:4 has 103 pQ repeats, and SEQ ID NO:6 has 25 pQrepeats. However, the numeric order of the amino acids 1-68 of Exon 1will not be changed by the number of pQ repeats. Furthermore, these andother pQ comprising polypeptides of the invention are contemplated tohave between 10 to 150 pQ repeats. This includes 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,132, 133, 134, 135, 136, 1317, 13, 139, 140, 141, 142, 143, 144, 145,146, 147, 148, 149, 150 pQ repeats.

F. Biological Functional Equivalents

One can also modify the sequence of any protein involved in fibrilformation and/or in protein aggregation; or a heat shock protein; or amolecular chaperone protein, by amino-acid substitutions, replacements,insertions, deletions, truncations and other mutations to obtain fibrilinhibitory and/or disassembling properties. These modification cangenerate functionally equivalent polypeptides may be obtained. Thefollowing is a discussion based upon changing of the amino acids of aprotein or polypeptide to create an equivalent, or even an improved,second-generation molecule. For example, certain amino acids may besubstituted for other amino acids in a protein structure withoutappreciable loss of interactive binding capacity with structures suchas, for example, antigen-binding regions of antibodies or binding siteson substrate molecules. Since it is the interactive capacity and natureof a protein that defines that protein's biological functional activity,certain amino acid substitutions can be made in a protein sequence, andin its underlying DNA coding sequence, and nevertheless produce aprotein with like properties (see Table 4). It is thus contemplated bythe inventors, that various changes may be made in the polypeptidesequences of the proteins involved in fibril formation and/or in proteinaggregation; or a heat shock protein; or a molecular chaperone protein,with no change in the normal activity of the polypeptide.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte & Doolittle, 1982). It is accepted that therelative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like.

It also is understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein. As detailed in U.S. Pat. No. 4,554,101, thefollowing hydrophilicity values have been assigned to amino acidresidues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate(+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine(0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine*−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine(−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5);tryptophan (−3.4).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still produce a biologicallyequivalent and immunologically equivalent protein. In such changes, thesubstitution of amino acids whose hydrophilicity values are within ±2 ispreferred, those that are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions generally are based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take into consideration the variousforegoing characteristics are well known to those of skill in the artand include: arginine and lysine; glutamate and aspartate; serine andthreonine; glutamine and asparagine; and valine, leucine and isoleucine.TABLE 4 Codon Table Amino Acids Codons Alanine Ala A GCA GCC GCG GCUCysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu EGAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGUHistidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAAAAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCAUCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUUTryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

Another embodiment for the preparation of polypeptides or proteininvolved in fibril formation and/or in protein aggregation; or a heatshock protein; or a molecular chaperone protein, is the use of peptidemimetics. Mimetics are peptide-containing molecules that mimic elementsof protein secondary structure. The underlying rationale behind the useof peptide mimetics is that the peptide backbone of proteins existschiefly to orient amino acid side chains in such a way as to facilitatemolecular interactions, such as those of antibody and antigen. A peptidemimetic is expected to permit molecular interactions similar to thenatural molecule.

G. Fusion Proteins

A fusion protein or chimeric protein is a specialized kind of proteinvariant that is an insertional variant. This molecule generally has allor a substantial portion of the native molecule, linked at the N- orC-terminus or in even at other parts of the protein, to all or a portionof a second polypeptide. In the present invention, fusion proteins havebeen generated that comprise regions/portions of the proteins involvedin fibrinl formation and/or in protein aggregation; or a heat shockprotein; or a molecular chaperone protein, that can be identified usingeither a fluorescence measuring method, a screening assay, or afunctional assay. For example, fusions comprising a region of a alphasynuclein protein, a huntingtin protein, etc. linked to a greenfluorescent protein (GFP) are described. Some of the GFP chimeras areN-terminal chimeras. Almost any type of fusion/chimeric protein may beprepared where the GFP region may be linked to other parts of theprotein of interest. Other useful chimeras include linking of functionaldomains, such as active sites from enzymes, or epitopes that can berecognized by antibodies. These fusion proteins provide methods forrapid and easy detection and identification of the recombinant hostcell, exemplified herein by the yeast cell.

H. Screening Methods of the Invention

The present invention provides methods for screening for candidatesubstances that prevent the misfolded protein disease and/or proteinfibrillogenesis and/or the accumulation of protein deposits in tissues.In some embodiments these agents prevent protein misfolding.Irrespective of the exact mechanism of action, agents identified by thescreening methods of the invention will provide therapeutic benefit todiseases involving protein misfolding or aberrant protein deposition.Some of these disorders are listed in Table 1 and include asnon-limiting examples, neurodegenerative diseases such as, Huntington's,Parkinson's, Alzheimer's, prion-diseases, etc. as well as othernon-neuronal diseases for example, type 2 diabetes.

The screening methods of the invention use yeast cells that areengineered to express proteins involved in fibril formation and/or inprotein aggregation. The yeast cell also requires one of the twoconditions described below for the screening method. In one module, theyeast cell have a mutant genetic background, for example, mutations inHSP genes or other molecular chaperone encoding genes, such as mutationsin the HSP40 gene. Alternatively, the yeast cell expressing a proteininvolved in fibril formation and/or in protein aggregation can besubject to changes in growth conditions that lead to stress, such asoxidative stress, for example by exposing the cell to a free radicalgenerator, or iron etc. Either of these conditions confers a toxicphenotype on the yeast cells expressing proteins involved in fibrilformation and/or in protein aggregation. Contacting such a yeast cellwith a candidate substance allows the identification of agents that canrescue the toxic phenotype of the yeast cell. The toxic phenotype ismanifested as cytotoxicity or growth inhibition. The toxicity in yeastcorrelates to the cytotoxic effect of the protein in a human cell thatcauses the pathology associated with the disease caused by proteinaccumulation. For example, the expression of the huntingtin protein in ayeast cell, which additionally has a mutant HSP40 background, makes theyeast cell severely growth retarded. Contacting such yeast cells withcandidate substances allows identification of agents that can reversethe growth retardation of yeast cells and hence the agent should alsoprevent the accumulation of huntingtin in a human cell. As huntingtinaggregation is involved in Huntington's disease, this screening methodprovides therapeutic agents to prevent and treat Huntington's disease.

(a) Candidate Substances

A “candidate substance” as used herein, is any substance with apotential to reduce, alleviate, prevent, or reverse theaccumulation/aggregation of proteinaceous deposits in tissues. Varioustype of candidate substances may be screened by the methods of theinvention. Genetic agents can be screened by contacting the yeast cellwith a nucleic acid construct encoding for a gene. For example, one mayscreen cDNA libraries expressing a variety of genes, to identifytherapeutic genes for the diseases described herein. In other examplesone may contact the yeast cell with other proteins or polypeptides whichmay confer the therapeutic effect.

Thus, candidate substances that may be screened according to the methodsof the invention include those encoding chaperone molecules, heat shockproteins, receptors, enzymes, ligands, regulatory factors, andstructural proteins. Candidate substances also include nuclear proteins,cytoplasmic proteins, mitochondrial proteins, secreted proteins,plasmalemma-associated proteins, serum proteins, viral antigens,bacterial antigens, protozoal antigens and parasitic antigens. Candidatesubstances additionally comprise proteins, lipoproteins, glycoproteins,phosphoproteins and nucleic acids (for example, RNAs such as ribozymesor antisense nucleic acids). Proteins or polypeptides which can bescreened using the methods of the present invention include chaperoneproteins, hormones, growth factors, neurotransmitters, enzymes, clottingfactors, apolipoproteins, receptors, drugs, oncogenes, tumor antigens,tumor suppressors, structural proteins, viral antigens, parasiticantigens and bacterial antigens. In addition, numerous methods arecurrently used for random and/or directed synthesis of peptide, andnucleic acid based compounds. The nucleic acid or protein sequencesinclude the delivery of DNA expression constructs that encode them.

In addition, candidate substances can be screened from large librariesof synthetic or natural compounds. One example, is a FDA approvedlibrary of compounds that can be used by humans. In addition, syntheticcompound libraries are commercially available from a number of companiesincluding Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex(Princeton, N.J.), Brandon Associates (Merrimack, N.H.), and Microsource(New Milford, Conn.) and a rare chemical library is available fromAldrich (Milwaukee, Wis.). Combinatorial libraries are available and canbe prepared. Alternatively, libraries of natural compounds in the formof bacterial, fungal, plant and animal extracts are also available, forexample, Pan Laboratories (Bothell, Wash.) or MycoSearch (N.C.), or canbe readily prepared by methods well known in the art. It is proposedthat compounds isolated from natural sources, such as animals, bacteria,fungi, plant sources, including leaves and bark, and marine samples maybe assayed as candidates for the presence of potentially usefulpharmaceutical agents. It will be understood that the pharmaceuticalagents to be screened could also be derived or synthesized from chemicalcompositions or man-made compounds.

Other suitable modulators include antisense molecules, ribozymes, andantibodies (including single chain antibodies), each of which would bespecific for the target molecule. Such compounds are described ingreater detail elsewhere in this document. For example, an antisensemolecule that bound to a translational or transcriptional start site, orsplice junctions, would be ideal candidate inhibitors. Additionally,natural and synthetically produced libraries and compounds are readilymodified through conventional chemical, physical, and biochemical means.

Screening of such libraries, including combinatorially generatedlibraries (e.g., peptide libraries), is a rapid and efficient way toscreen large number of related (and unrelated) compounds for activity.Combinatorial approaches also lend themselves to rapid evolution ofpotential drugs by the creation of second, third and fourth generationcompounds modeled of active, but otherwise undesirable compounds.

Useful compounds may be found within numerous chemical classes, thoughtypically they are organic compounds, including small organic compounds.Small organic compounds have a molecular weight of more than 50 yet lessthan about 2,500 daltons, preferably less than about 750, morepreferably less than about 350 daltons. Exemplary classes includeheterocycles, peptides, saccharides, steroids, triterpenoid compounds,and the like. Structural identification of an agent may be used toidentify, generate, or screen additional agents. For example, wherepeptide agents are identified, they may be modified in a variety of waysto enhance their stability, such as using an unnatural amino acid, suchas a D-amino acid, particularly D-alanine, by functionalizing the aminoor carboxylic terminus, e.g. for the amino group, acylation oralkylation, and for the carboxyl group, esterification or amidification,or the like.

(b) Screen with FRET and FACS

In one embodiment, the invention contemplates screening assays usingfluorescent resonance energy transfer (FRET). In one example,alpha-synuclein is fused to cyan fluorescent protein (CFP) and to yellowfluorescent protein (YFP) and is integrated in the yeast genome underthe regulation of a Gall-10 promoter. Cells are grown in galactose toinduce expression. Upon induction, cells produce the fusion proteins,which aggregate bringing the CFP and YFP close together. Becauseproteins in the aggregates are tightly packed, the distance between theCFP and YFP is less than the critical value of 100° A that is necessaryfor an energy transfer (FRET) to occur. In this case, the energyreleased by the emission of CFP will excite the YFP, which in turn willemit at its characteristic wavelength. The present inventors contemplateutilizing FRET bases screening to identify candidate compoundsincluding, drugs, genes or other factors that can disrupt theinteraction of CFP and YFP by maintaining the proteins in a state thatdoes not allow aggregation to occur.

Cells will be sorted by fluorescence activated cell sorting (FACS)analysis, a technique well known to those of skill in the art. Theinventors envision that this method of screening also enables theinvestigation of toxic intermediates formed in the aggregation pathwayand will eventually allow a better understanding of how intermediatesaggregate into insoluble proteins often characterized by plaques andtangles.

FACS, flow cytometry or flow microfluorometry provides the means ofscanning individual cells for the presence of fluorescentlylabeled/tagged moiety. The method employs instrumentation that iscapable of activating, and detecting the excitation emissions of labeledcells in a liquid medium. FACS is unique in its ability to provide arapid, reliable, quantitative, and multiparameter analysis on eitherliving or fixed cells. The misfolded disease proteins of the presentinvention, suitably labeled, provide a useful tool for the analysis andquantitation of protein aggregation and fibril and/or aggregateformation as a result of other genetic or growth conditions ofindividual yeast cells as described above.

(c) RNA Aptamers Screen

In another embodiment, the invention contemplates screening assays usingRNA-aptamers. RNA is a nucleic acid capable of adopting a vast number ofsecondary structures, depending on its primary sequence. It is thereforepossible to engineer RNA molecules with specific lengths so that theyhave the property of binding other molecules in a very specific mannerand with very high affinity. This is similar to the phenomenon ofantigen-antibody association.

The present inventors contemplate utilizing these properties of RNAmolecules to identify RNA molecules that are candidate therapeuticagents for protein misfolding diseases, such as the neurodegenerativediseases. This is based on the ability of RNA molecules to recognize andbind misfolded disease proteins for example the amyloid fibers or otherintermediate species in the pathway of aggregate/fibril formation.

The yeast-based screening system developed herein is amenable to suchscreens, and one may directly identify compounds that decrease thetoxicity of misfolded disease proteins. In addition, one may identifycompounds that disrupt the interaction between intermediates formed thatlead to the aggregation of such proteins. It is also contemplated thatone can screen for compounds that aggravate the toxicity or promoteprotein aggregation.

(d) Treatments

Initial testing and treatment of animal-models with test compoundsidentified by the screens of the invention are also contemplated.Suitable animal-model for the protein misfolding disease will beselected and treatment will involve the administration of the compound,in an appropriate pharmaceutical formulation, to the animal.Administration will be by any route that could be utilized for clinicalor non-clinical purposes, including but not limited to oral, nasal,buccal, or even topical. Alternatively, administration may be byintratracheal instillation, bronchial instillation, intradermal,subcutaneous, intramuscular, intraperitoneal or intravenous injection.Specifically-contemplated routes are systemic intravenous injection,regional administration via blood or lymph supply, or directly to anaffected site. Determining the effectiveness of a compound in vivo mayinvolve a variety of different criteria. Also, measuring toxicity anddose response can be performed in animals in a more meaningful fashionthan in in vitro or in cyto assays.

I. Immunological Detection

It is also contemplated that one may detect the misfolded diseaseprotein expression in the engineered yeast cells by immunologicalmethods using suitable anti-misfolded disease protein antibodies. Onecan also use anti-heat shock protein antibodies or other anti-chaperoneantibodies to detect the specific type of genetic mutation present in ayeast cell. The proteins, and/or polypeptides that can be detectedinclude mutated versions.

In still further embodiments, the present invention thus concernsimmunodetection methods for binding, purifying, removing, quantifying orotherwise generally detecting biological components. The steps ofvarious useful immunodetection methods have been described in thescientific literature, such as, e.g., Nakamura et al. (1987;incorporated herein by reference). Immunoassays, in their most simpleand direct sense, are binding assays. Certain preferred immunoassays arethe various types of enzyme linked immunosorbent assays (ELISAs),radioimmunoassays (RIA) and immunobead capture assay.Immunohistochemical detection using tissue sections also is particularlyuseful. However, it will be readily appreciated that detection is notlimited to such techniques, and Western blotting, dot blotting, FACSanalyses, and the like also may be used in connection with the presentinvention.

In general, immunobinding methods include obtaining a yeast celltransformed with an expression construct expressing a protein or peptideand contacting the sample with an antibody to the protein or peptide inaccordance with the present invention, as the case may be, underconditions effective to allow the formation of immunocomplexes.

The immunobinding methods of this invention include methods fordetecting or quantifying the amount of a reactive component in a sample,which methods require the detection or quantitation of any immunecomplexes formed during the binding process. Here, one would obtain ayeast cell transformed with an expression construct expressing a proteinor peptide of the invention and contact the sample with an antibody andthen detect or quantify the amount of immune complexes formed under thespecific conditions.

Contacting the chosen biological sample with the protein, peptide orantibody under conditions effective and for a period of time sufficientto allow the formation of immune complexes (primary immune complexes) isgenerally a matter of simply adding the composition to the sample andincubating the mixture for a period of time long enough for theantibodies to form immune complexes with, i.e., to bind to, any antigenspresent, such as antigens corresponding to misfolded disease proteins.After this time, the sample-antibody composition, such as a tissuesection, ELISA plate, dot blot or Western blot, will generally be washedto remove any non-specifically bound antibody species, allowing onlythose antibodies specifically bound within the primary immune complexesto be detected.

In general, the detection of immunocomplex formation is well known inthe art and may be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any radioactive, fluorescent, biological orenzymatic tags or labels of standard use in the art. U.S. patentsconcerning the use of such labels include U.S. Pat. Nos. 3,817,837;3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241,each incorporated herein by reference. Of course, one may findadditional advantages through the use of a secondary binding ligand suchas a second antibody or a biotin/avidin ligand binding arrangement, asis known in the art.

The encoded protein, peptide or corresponding antibody employed in thedetection may itself be linked to a detectable label, wherein one wouldthen simply detect this label, thereby allowing the amount of theprimary immune complexes in the composition to be determined.

Alternatively, the first added component that becomes bound within theprimary immune complexes may be detected by means of a second bindingligand that has binding affinity for the encoded protein, peptide orcorresponding antibody. In these cases, the second binding ligand may belinked to a detectable label. The second binding ligand is itself oftenan antibody, which may thus be termed a “secondary” antibody. Theprimary immune complexes are contacted with the labeled, secondarybinding ligand, or antibody, under conditions effective and for a periodof time sufficient to allow the formation of secondary immune complexes.The secondary immune complexes are then generally washed to remove anynon-specifically bound labeled secondary antibodies or ligands, and theremaining label in the secondary immune complexes is then detected.

Further methods include the detection of primary immune complexes by atwo step approach. A second binding ligand, such as an antibody, thathas binding affinity for the encoded protein, peptide or correspondingantibody is used to form secondary immune complexes, as described above.After washing, the secondary immune complexes are contacted with a thirdbinding ligand or antibody that has binding affinity for the secondantibody, again under conditions effective and for a period of timesufficient to allow the formation of immune complexes (tertiary immunecomplexes). The third ligand or antibody is linked to a detectablelabel, allowing detection of the tertiary immune complexes thus formed.This system may provide for signal amplification if this is desired.

J. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE 1 Materials and Methods for Ht Expression in Yeast

Plasmid Construction

Plasmids encoding fusions between GFP and the N-terminal region of Htwere the kind gift of the Hereditary Disease Foundation. To create yeastexpression plasmids for HtQ25 or HtQ103 DNAs were digested with Xho Iand Xba I, and the resulting Xho I/Xba I fragments were ligated into thevector pYES (Invitrogen) to obtain plasmids pYES/PQ25 or pYES/PQ103,respectively. These DNAs were digested with Sal I and ends were filledwith Klenow enzyme. Afterwards DNAs were digested with EcoR I and theresulting fragments were subcloned into a high copy (2μ) expressionvector p426 for constitutive expression or p426GAL for galactoseinduction, respectively (Mumberg et al., 1994; Mumberg et al., 1995).

To create low copy (CEN) expression plasmids with either constitutive(GPD) or galactose (GAL) inducible promotor DNAs, p426/PQ25 orp426/PQ103 were digested with Xho I. The resulting Xho I fragments weresubcloned into p416 or p416GAL, respectively (Mumberg et al., 1994;Mumberg et al., 1995).

To generate the same set of yeast expression plasmids for HtQ47 or HtQ72DNAs were double-digested with Acc 65I and Xba I, fragments were bluntedwith Klenow enzyme, and subcloned into a Cla I-blunted vector p426 forconstitutive expression. To generate low copy expression plasmids withconstitutive expression (GPD) DNA p426/PQ47 or p426/PQ72 were digestedwith Spe I and Xho I, and the resulting fragments were subcloned intop416.

The expression plasmids used in this study are listed in Table 5 (Kimuraet al., 1995; Nathan et al., 1995; Vogel, et al., 1995). TABLE 5Plasmids Used Plasmid Promoter Copy Number Reference p416 GPD CEN, lowMumberg, 1995 p416/PQ25 GPD CEN, low this study p416/PQ47 GPD CEN, lowthis study p416/PQ72 GPD CEN, low this study p416/PQ103 GPD CEN, lowthis study p426 GPD 2μ, high Mumberg, 1995 p426/PQ25 GPD 2μ, high thisstudy p426/PQ47 GPD 2μ, high this study p426/PQ72 GPD 2μ, high thisstudy p426/PQ103 GPD 2μ, high this study p416GAL GAL CEN, low Mumberg,1994 p416Gal/PQ25 GAL CEN, low this study p416Gal/PQ103 GAL CEN, lowthis study p426GAL GAL 2μ, high Mumberg, 1994 p426Gal/PQ25 GAL 2μ, highthis study p426Gal/PQ103 GAL 2μ, high this study pTVSIS1 GPD 2μ, highunpublished pRSYDJ1 GPD CEN, low Kimura et al., 1995 pLH101 GPD 2μ, highunpublished pTGpd/P82 GPD CEN, low Nathan & Lindquist, 1995 p2HG(104)GPD 2μ, high Vogel et al., 1995

Transformation of yeast was performed using a standard lithium/PEGmethod (Ito et al., 1983).

Yeast strains, Transformation and Cultivation

In this study we used five isogenic series of yeast strains, in thebackgrounds: W303 (MATa can1-100 ade2-1 his3-11, 15 trp1-1 ura3-1leu2-3, 112), YPH499 (MATa ade2-101ochre his3-Δ200 leu2-Δ1 lys2-801ambertrp-Δ63 ura3-52), MHY810 (MATa his3-Δ200 leu2-Δ1 lys1-1 met14ura3-Δ1::TRP1 trp1-Δ1), MHY501 (MATa his3-Δ200 leu2-3, 112 ura3-52lys2-801 trp1-1) and MHY803 (MHY501 derivative: MATa his3-Δ200 leu2-3,112 ura3-52 lys2-801 trp1-1 (doa3::HIS3+) (Ycplac22-Doa3-His₆)). The MHYstrains were kind gifts from Mark Hochstrasser. Yeast strains used arelisted in Table 6. TABLE 6 Aggregation of Mutant Huntingtin in DifferentYeast Strains 2μ plasmids CEN plasmids Aggregation of Aggregation of Q25Q47 Q72 Q103 Q25 Q47 Q72 Q103 Strain MHY810 wild-type − −/+ + ++ MHY898sen3-1 − −/+ + ++ − + ++ MHY803 wild-type − −/+ + ++ MHY792 doa3-1 −−/+ + ++ − + ++ MHY501 wild-type − −/+ + ++ MHY1408 uba1 − −/+ + ++YPH499 wild-type − −/+ + ++ DYJ1 Δydj1 − −/+ + ++ W303 wild-type − −/+ +++ − −/+ + ++ LP6-2 Δhsp26 − −/+ + ++ LP8-1 Δhsp35 − −/+ + ++ SL314-A1Δssa1ssa2 − −/+ + ++ + ++ SL318-2A Δssa3ssa4 − −/+ + ++ CLD82a Δhsc82 −−/+ + ++ + ++ iLEP1α Δhsc82 − −/+ + ++ SL304A Δhsp104 − − − − − − − −Overexpression wild-type YDJ1 − + ++ wild-type SIS1 − + ++ wild-typeSSA1 − + + wild-type HSP82 − + ++ wild-type HSP104 − + + Δhsp104 YDJ1 −− − Δhsp104 SIS1 − − −Empty space no transformation made− no foci fluorescence−/+ a minority of cells have one small focus+ one or more foci, with considerable background fluorescence++ one or two intense fluorescence foci, with lower backgroundfluorescence

Transformation of yeast was performed using a standard lithium/PEGmethod (Ito et al., 1983).

Yeast cells were grown in rich media (YPD) or in minimalglucose/raffinose/galactose medium (Adams et al., 1997) deficient forthe required amino acids for plasmid selection. For experimentalpurposes cells were grown overnight at 25° C. into log, late-log orearly stationary phase.

Sedimentation Analysis

Yeast cells were harvested by centrifugation at 1500×g for 5 min at roomtemperature and washed once in 10 mM ethylenediaminetetraacetic acid(EDTA). Cells were resuspended in spheroplasting buffer (1 M sorbitol,0.1 M EDTA, 0.5 mg/ml zymolyase 100T (Seikagaku Corporation), 50 mMdithiothreitol, pH 7.5) and incubated for 2 h at 30° C. Afterwards,spheroplasts were harvested by mild centrifugation at 325×g for 5 min at4° C. and lysed in 1×TNE containing a protease-inhibitor cocktail(complete Mini-tablets, Boehringer Mannheim). After incubation in1×TNE+2% Sarcosyl for 5 mm on ice, samples were loaded onto a 5% (w/v)sucrose cushion (1 M sucrose, 100 mM NaCl, 0.5% sulfobetaine) andcentrifugation was performed at 315,000×g for 1 hr at 4° C. Afterwards,supernatant and pellet fractions were subjected to 8% SDS-PAGE (Novex)and transferred to a polyvinylidene fluoride membrane (MilliporeCorporation). Membranes were blocked with 5% nonfat-dehydrated milkpowder in phosphate buffered saline (PBS) for 1 hr. Incubation with theprimary antibody was performed overnight at 4° C. After incubation withProtein A-peroxidase (1:5000, Boehringer Mannheim), the immune complexeswere visualized by treating membranes with ECL reagent (Amersham).Antibody UGFP was used at 1:100 (Clontech).

Microscopy

Yeast cells were allowed to adhere onto polylysine-treated slides for 10min. For nucleus staining, cells were fixed with 1% formaldehyde for 5min and washed 3 times with PBS. After treatment with4′,6-diamidine-2-phenylindole-dihydrochloride (DAPI, Sigma) for 5 min,cells were washed 3 times with PBS. Microscopy was performed with aAxioplan 2 microscope (Zeiss), and micrographs were taken at amagnification of 100×.

EXAMPLE 2 Coalescence of Mutant Ht in Yeast

To investigate Ht in yeast, the N-terminal region (amino acids 1-68 ofthe wild-type protein) with a wild-type polyQ (polyglutamine) repeatlength of 25 residues or with mutant repeat lengths of 47, 72 or 103residues was fused to GFP. Each was placed under the control of GPD, astrong constitutive yeast promoter, on a single copy plasmid (FIG. 1).Homopolymeric tracts of CAG, the naturally occurring glutamine codon inHt, are inherently unstable, and particularly so in yeast (Moore et al.,1999; Schweitzer et al., 1997). This problem was reduced by the factthat glutamine is encoded by both CAG and CAA and that mixed codonrepeats are considerably more stable (Kazantsev et al., 1999). Tominimize instability problems, all experiments reported herein wereperformed with mixed codon polyQ repeats and all work was performed withfresh transformants, using at least two independent colonies in eachcase, and repeated at least two times.

Fluorescence from GFP-fusion proteins containing wild-type polyglutaminetracts (25 residues; HtQ25) was always distributed diffusely throughoutthe cell (FIG. 1, middle). HtQ47 fluorescence was also diffuselydistributed, although coalescent foci were observed in a smallpercentage of cells (less than 2%). More than half of cells expressingHtQ72 exhibited a single intense spot of fluorescence against a diffusefluorescent background. Virtually all cells expressing HtQ103 exhibiteda single intense spot of fluorescence, with much less backgroundfluorescence than seen with other variants. When the same constructswere expressed from high-copy plasmids (p426 series, Table 5),fluorescence intensity was much greater, but the pattern of fluorescencewas very similar. Immunoblotting of total cellular protein indicatedthat all four variants were expressed at similar levels. Thus, thedegree of coalescence exhibited by the N-terminal fragment of Ht dependsmore upon the length of the polyglutamine tract than the level ofprotein expressed.

EXAMPLE 3 Newly Induced Mutant Ht Aggregates in All Cells

Cells expressing different Q repeat variants exhibited the samefrequency of plasmid loss (determined by plating cells to non-selectiveand selective media) and grew at similar rates, with only a slightdeficit in cells expressing HtQ103. Final densities were typically0.7-0.8 for cells expressing HtQ25, HtQ47 or HtQ72 and 0.5-0.7 for cellsexpressing HtQ103. Thus, the long polyQ Ht fragments were not overtlytoxic in yeast. However, because the proteins were expressed from aconstitutive promoter, it was possible that a subset of cells competentto grow in the presence of polyQ proteins had been selected duringtransformation. If so, selection might also have influenced theaggregation state of the proteins. To determine if the coalescence ofexpanded glutamine reflected an inherent property of the protein or wasthe result of a selective process, the Ht-GFP constructs weretransferred to the control of a galactose-inducible promoter (Table 5).Transformants were selected on glucose plates to keep the constructtightly repressed. To initiate induction, cells were first grown inraffinose medium overnight, to eliminate glucose repression, and thentransferred to galactose medium, to induce Ht expression.

Bright GFP fluorescence was observed after 4 hours, but for all threevariants tested, HtQ25, HtQ72, and HtQ103, fluorescence was diffuselydistributed. With continued expression HtQ72 and HtQ103 coalescencebegan to appear in some cells after 9 hrs (2 doublings). After 24 hrs,coalescence was indistinguishable from that observed in culturesexpressing the Ht variants constitutively and all cultures had reachedsimilar densities. Thus, coalescence of expanded glutamine repeatsoccurs in most, if not all, cells in the culture, but many hours ofexpression are required for it to occur.

EXAMPLE 4 Mutant Ht Forms Cytoplasmic Aggregates in Yeast

Co-staining cells with DAPI, a DNA-binding dye that fluoresces blue,demonstrated that foci of Ht coalescence were in the cytoplasmiccompartment not in the nucleus. To determine if these foci reflected thesequestration of Ht-GFP fusions into a membrane-bounded compartment orthe formation of higher order protein complexes, cell walls were removedand cells were lysed in the presence of the detergent Sarkosyl (2%).After sedimentation, supernatant and pellet fractions were boiled insample buffer containing 5% SDS for 10 minutes and analyzed byimmunoblotting.

HtQ25 and HtQ47 were detected only in supernatant fractions. HtQ72 wasdistributed between supernatant and pellet fractions, whereas virtuallyall HtQ103 protein was found in the pellet fraction. Note that afterelectrophoresis a major fraction of HtQ103 remained at the top of thegel. Apparently, the coalescence detected through GFP fluorescence wasdue to the formation of higher order complexes. For HtQ103, and less sofor HtQ72, these complexes resisted solubilization by boiling in 5% SDS.

EXAMPLE 5 Aggregates are Unaltered in Proteasome-Deficient Cells

Because aggregates of Ht (Saudou et al., 1998) and otherglutamine-repeat proteins associated with disease, such as SBMA(Stenoien et al., 1999), SCA3 (Chai et al., 1999) and SCA1 Cummings etal., 1998), are ubiquitinated in mammalian cells and are associated withcomponents of the proteosome, it has been suggested that theubiquitin/proteosome pathway might be involved in aggregate formation.Ubiquitinated Ht proteins were not detected in yeast cells. However,even for proteins known to be turned over by this pathway ubiquitinconjugates can be difficult to detect. To investigate this question morerigorously, a genetic approach was undertaken. Three strains wereemployed, each containing a lesion in a different component of theubiquitin/proteasome degradation pathway: 1) uba1, the ubiquitinactivating enzyme (M. Hochstrasser), 2) doa3, a catalytic subunit of the20S proteasome (Chen et al., 1995), and 3) sen3 a subunit of the 19Sproteasome regulatory complex (DeMarini et al., 1995). Since each ofthese genes is essential, partial loss-of-function mutations were usedthat severely impair this pathway. In each of the strains, the Htvariants behaved in the same manner as they did in wild-type cells.There were no changes in the number of cells containing coalescent foci,nor in the size or intracellular distribution of those foci (Table 6).

EXAMPLE 6 Molecular Chaperones Affect Aggregation of Ht

Chaperone proteins are a highly conserved, but diverse group of proteinsthat control the folding of other proteins by interacting with differenttypes of folding intermediates and off pathway folding products(Gething, 1997). They have profound effects on the aggregation ofabnormal proteins. To determine how changes in the levels of chaperoneproteins would affect the coalescence of the Ht polyQ variants, anisogenic series of strains was generated containing deletion mutationsor over-expression plasmids for various chaperone proteins, whichproduced wild-type or polyQ expanded Ht fragments. Note that somechaperone deletions could not be tested because they are lethal.

Most of the tested alterations in chaperone proteins had no noticeableeffects on the intracellular distribution of Ht variants as determinedby GFP fluorescence (Table 6) and no significant effect on the manner inwhich the Ht fragments were partitioned between the supernatant andpellet fractions after sedimentation. This category included mutationsthat eliminated the expression of the major small Hsp (in yeast, Hsp26)(Petko et al., 1986), 2) increased the expression of Hsp90 (in yeast,Hsc/p82) several fold (Borkovich et al., 1989) or reduced the expressionof Hsp90 by 10- to 15-fold (Nathan et al., 1999), 3) eliminated theexpression of various members of the essential cytosolic Hsp70 family(constitutive members Ssa1 and Ssa2 (Parsell et al., 1994a and b), andstress inducible members Ssa3 and Ssa4, and 4) increased or eliminatedexpression of Ydj1 (a member of the Hsp40 family) (Kimura et al., 1995).A deletion of Hsp35 was also examined. This heat-inducible protein is amember of the glyeraldehyde-3-phosphate dehydrogenase family and ispostulated to be a chaperone because it is both heat inducible and oneof the most abundant proteins in yeast (Boucherie et al., 1995).Mammalian glyeraldehyde-3-phosphate dehydrogenase exhibits a glutaminelength-dependent association with Ht (Burke et al., 1996).

Over-expression of three chaperones had significant effects. Sis1, amember of the Hsp40 family, caused two intense foci of aggregation toappear in most cells with HtQ72 and HtQ103, rather than the single focusof coalescence observed in virtually all wild-type cells. In cellsover-expressing Hsp70 (Ssa1), HtQ72 and HtQ103 fluorescence was muchmore variable than in wild-type cells. Multiple foci of fluorescencewere observed in many cells, and many also contained a higher backgroundof diffuse fluorescence. This variability likely reflects differences inplasmid copy number, which is commonly observed with Hsp70 expressionplasmids (Stone et al., 1990). Over-expression of Hsp104 also increasedthe number of fluorescent foci and the background fluorescence observedwith the Ht variants HtQ72 and HtQ103 (Table 6). It also increased therelative quantities of HtQ72 and HtQ103 found in the supernatantfractions after centrifugation. Curiously, although HtQ72 proteinappeared at least partially aggregated in these cells, little proteinfractionated in the pellet in three out of three experiments. Theprotein may be more loosely packed or in a Sarkosyl-soluble state.

Of all the chaperone alterations tested, a deletion of the HSP104 genehad the most dramatic effect. In these strains, all of the Ht variantfragments exhibited diffuse fluorescence. The same results were obtainedwith both the high and low copy Ht expression constructs (Table 6).Moreover, by sedimentation, all of the proteins were only detected insupernatant fractions.

EXAMPLE 7 Hsp40 Alters Aggregation of Ht Variants

That Sis1 (a class II yeast Hsp40 protein) affected the aggregationstate of huntingtin and that Hsp40 proteins, most particularly HDJ-1,seem to play a crucial role in polyQ-induced toxicity in several modelsystems led to a more detailed analysis of Sis1. Yeast strainsengineered to express different regions of the Sis1 protein weretransformed with the huntingtin-GFP fusion constructs described in theabove Examples. The aggregation pattern of HtQ72 and HtQ103 was markedlyaltered by the production of mutant Sis1 proteins. Instead of a smallnumber of large aggregates, a large number of smaller aggregates werepresent. Most notably, with one Sis1 construct this change inaggregation was accompanied by a reduction in viability. ThisSis1-induced toxicity can be reduced by co-expression of Hsp104. Hsp04also reduces both aggregate formation and cell death in a mammalian cellmodel of huntingtin toxicity and in a C. elegans model employing simplepolyQ-GFP-fusions. These striking observations suggest that the toxicityof huntingtin induced by Sis1 alterations in yeast can be considereddirectly related to its toxicity in humans.

EXAMPLE 8 Compounds that Affect Huntingtin Toxicity in Yeast

Using the strain expressing the truncated Sis1 protein and HtQ103(toxic), screens were performed using the spotting assays described inExamples 9 and 10 below. Further screens with other candidate agents arealso contemplated. These screens allow identification of agents thataffect huntingtin aggregation in the living cell but are by themselvesnon-toxic.

Specifically, yeast strains in the mutant Sis1 background expressingHtQ25 (control, not toxic), or HtQ72 (not toxic, but potentially so), orHtQ103 (toxic) were spotted in serial dilutions onto selective mediawith or without test compounds. HtQ47 will also be spotted in serialdilutions onto selective media with or without test compounds in similarexperiments. Increased growth rate on test plates compared to controlplates identifies compounds with a potential for reducing toxicity; adecreased growth identifies compounds that might increase toxicity.Microscopic analysis determines whether these agents also affectaggregate formation by the GFP-fusion proteins. This screen will also beperformed with yeast strains expressing only the N-terminal region ofhuntingtin, not fused to GFP.

Spotting assays demonstrate that various compounds, including severallisted in Table 3, induce toxicity in the yeast strains which compriseW303, hsp104, and Sis1 mutants.

One concern in yeast screens is that some agents may not be able toenter the yeast cell, can not be taken up, are rapidly metabolized, orpumped out of the cell. To eliminate this possibility a variant of yeaststrains designed to eliminate this problem will be employed. A strainmutant in 3 genes (erg6, pdr1, and pdr3) affecting membrane efflux pumps(Cummings et al., 1998) and increasing permeability for drugs (Chen etal., 1995) will be used in initial studies. These particular strains hasbeen used very successfully in cancer research to identify growthregulators (see Website: http://dtp.nci.nih.gov).

EXAMPLE 9 Materials and Methods for Toxicity in Yeast ExpressingMisfolded Disease Proteins

Plasmid Constructions

Wild-type (WT), A53T and A30P alpha-synuclein cDNAs were a kind giftfrom Dr. Peter Lansbury. WT, A53T, and A30P sequences were subclonedinto p426GPD, p416GPD, p423GPD and p425GPD (Mumberg et al., 1995) bystandard molecular biology procedures. GFP, CFP and YFP fusions whichare fusions of alpha-synuclein in frame with GFP, CFP or YFP wereconstructed by inserting the XFP (X meaning G, C or Y) coding sequencein frame with alpha-synuclein in the same vectors. The XFP fusions werealso subcloned into pRS306 and pRS304 under the regulation of a GAL1-10promoter and with a Cyc1 terminator region.

Yeast Techniques

Yeast strains were grown and manipulated following standard procedures(see Guthrie and Fink, Guide to Yeast Genetics and Molecular Biology,Academic Press, 1991).

Spotting Experiments

Yeast cells were routinely grown overnight at 30° C. or at roomtemperature in selective media until they reached log or late log phase.Cells were counted using a hemocytometer and diluted to 1×10⁶ cells/ml.Five serial dilutions (five-fold) were made and cells were spotted ontomedia containing chemicals/drugs to screen.

EXAMPLE 10 Alpha-Synuclein Forms Fluorescent Foci in the Yeast Cytoplasm

Using GFP fusions the formation of fluorescent foci dispersed throughoutthe cytoplasm were detected. These inclusions were more prominent withthe WT and A53T mutant than with the A30P. Using this kind of assay theformation of inclusions by A30P cannot be ruled out, but a differentkind of aggregates has to be expected in this case, as the GFPfluorescence pattern looks different.

EXAMPLE 11 Conditions Increasing the Toxicity of Alpha-Synuclein

Using spotting experiments different categories of chemicals wereidentified as agents capable of aggravating the toxicity ofalpha-synuclein overexpression or of inducing toxicity of huntingtinexpression. Of the compounds tested in Table 3, the following had anegative effect on the growth of yeast cells overexpressing WTalpha-synuclein: carbon sources (arabinose 2%); nitrogen sources (urea 1mg/ml); salts and metals (CaCl₂ 0.5 M, CoCl₂ 750 μM, CsCl 0.1 M, CuSO₄2.5 mM, CuSo₄ 5 mM, Fe₂(SO₄)₃ 8.5 mM, FeSO₄ 20 mM, FeCl₂ 10 mM, FeCl₂ 15mM, FeCl₂ 23 mM, FeCl₂ 50 mM, MgCl₂ 0.5 M, MgSO₄ 0.5 M, RbCl 0.2 M,SrCl₂ 0.5 M); and, general inhibitors (6-azauracil 30 μg/ml,aurintricarboxylic acid 100 μM, bleomycin 1 μg/ml, brefeldin A 100μg/ml, camptothecin 5 μg/ml, chlorambucil 3 mM, ethidium bromide 50μg/ml, formamide 2%, GuHCl 20, hydroxyurea 5 mg/ml, menadione 20-50 μM,paraquat 1 mM (methyl viogen), vanadate 1 mM, vanadate 0.1 mM, vanadate2 mM, vanadate 4 mM, vanadate 7 mM+KCl.

Some compounds, in a preliminary study, exhibited an ability toalleviate toxocity caused by alpha-synuclein overexpression: Theseincluded: nitrogen source (serine 1 mg/ml); general inhibitors(camptothecin 0.1 μg/ml, DL-C-allylglycine 0.025 mg/ml, Hygromycin B 50μg/ml, L-ethionine 1 μg/ml, paromomycin 200 μg/ml, protamine sulphate250 μM); vitamins (B12); proteasome inhibitors (chloroquine 4.2 μM,clioquinol 5 μM, (R)-(−)-3-hydroxybutirate, L-DOPA); amyloid-relatedcompounds (Congo Red 5 μM, chrysamine G 1.0 μM, Deoxycorticosterone);and, anti-oxidants (glutathione).

EXAMPLE 12 Screen with FRET and FACS

Alpha-synuclein fused to CFP and to YFP was integrated in the yeastgenome under the regulation of a Gall-10 promoter (FIG. 4). Cells aregrown in galactose to induce expression. Upon induction, cells willproduce the fusion proteins, which will aggregate bringing the CFP andYFP close together. Because the proteins in the aggregates are tightlypacked, the distance between the CFP and YFP will be less than thecritical value of 100 ç that is necessary for an energy transfer (FRET)to occur. In this case, the energy released by the emission of CFP willexcite the YFP, which in turn will emit at its characteristicwavelength. Thus, this phenomenon can be used to identify drugs, genesor other factors that may disrupt this interaction by maintaining theproteins in a state that does not allow for aggregation to occur. Thesefactors will be analyzed by sorting cells by FACS analysis. This allowsthe investigation of toxic intermediates in the aggregation pathway andthus addresses whether the aggregates or other intermediates are causingcell death.

EXAMPLE 13 RNA Aptamers Screen

RNA aptamers will be screen to identify ones that would have potentialapplications as therapeutics for neurodegenerative diseases due to theirability to recognize and bind amyloid fibers or other intermediatespecies in the pathway. The yeast system would thus be very amenable fordoing this screens, either by looking directly for molecules that woulddecrease the toxicity of alpha-synuclein overexpression as well as bylooking for molecules that would disrupt the interaction between thespecies that lead to aggregation of the protein. Also interesting wouldbe to find some molecules that would aggravate the toxicity or promotethe aggregation, as this could give insight into the epitopes necessaryfor fibrillogenesis/aggregation of this and other proteins implicated inhuman disease.

All of the COMPOSITIONS and/or METHODS disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe COMPOSITIONS and/or METHODS and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1-68. (canceled)
 69. A method of screening for a compound that decreasestau associated toxicity, the method comprising: providing a yeast cellengineered to express a protein comprising a tau polypeptide; contactingthe yeast cell with a candidate compound; and evaluating the yeast cellfor viability, wherein an increase in viability of the yeast cell ascompared to viability of the yeast cell in the absence of the candidatecompound indicates that the candidate compound decreases tau associatedtoxicity.
 70. The method of claim 69, wherein the yeast cell has agenetic background that causes the yeast cell to have a reduced growthrate or no growth at all as a result of expressing the polypeptidecomprising tau.
 71. The method of claim 69, wherein the candidatecompound is a small molecule or a nucleic acid.
 72. A method ofscreening for a compound that decreases tau associated toxicity, themethod comprising: contacting a yeast cell with a candidate compound,wherein the yeast cell expresses a protein comprising a tau polypeptide;contacting the yeast cell with a toxicity inducing agent; and evaluatingthe yeast cell for viability, wherein an increase in viability of theyeast cell as compared to viability of the yeast cell in the absence ofthe candidate compound indicates that the candidate compound decreasestau associated toxicity.
 73. The method of claim 72, wherein thetoxicity inducing agent is a carbon source, nitrogen source,chemotherapeutic agent, alcohol, translation inhibitor, NSAID, DNAintercalator, chelator, liposome, antibiotic, vitamin, proteasomeinhibitor, anti-oxidant, or reducing agent.
 74. The method of claim 72,wherein the toxicity inducing agent is a metal or salt.
 75. The methodof claim 72, wherein the toxicity inducing agent is a compound thatcauses oxidative stress.
 76. The method of claim 75, wherein thecompound that causes oxidative stress is menadione or diamide.
 77. Themethod of claim 72, wherein the candidate compound is a small moleculeor a nucleic acid.